Isolated human kinase proteins, nucleic acid molecules encoding human kinase proteins, and uses thereof

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the kinase peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the kinase peptides, and methods of identifying modulators of the kinase peptides.

FIELD OF THE INVENTION

The present invention is in the field of kinase proteins that are related to the membrane-associated guanylate kinase subfamily, recombinant DNA molecules, and protein production. The present invention specifically provides novel peptides and proteins that effect protein phosphorylation and nucleic acid molecules encoding such peptide and protein molecules, all of which are useful in the development of human therapeutics and diagnostic compositions and methods.

BACKGROUND OF THE INVENTION

Protein Kinases

Kinases regulate many different cell proliferation, differentiation, and signaling processes by adding phosphate groups to proteins. Uncontrolled signaling has been implicated in a variety of disease conditions including inflammation, cancer, arteriosclerosis, and psoriasis. Reversible protein phosphorylation is the main strategy for controlling activities of eukaryotic cells. It is estimated that more than 1000 of the 10,000 proteins active in a typical mammalian cell are phosphorylated. The high energy phosphate, which drives activation, is generally transferred from adenosine triphosphate molecules (ATP) to a particular protein by protein kinases and removed from that protein by protein phosphatases. Phosphorylation occurs in response to extracellular signals (hormones, neurotransmitters, growth and differentiation factors, etc), cell cycle checkpoints, and environmental or nutritional stresses and is roughly analogous to turning on a molecular switch. When the switch goes on, the appropriate protein kinase activates a metabolic enzyme, regulatory protein, receptor, cytoskeletal protein, ion channel or pump, or transcription factor.

The kinases comprise the largest known protein group, a superfamily of enzymes with widely varied functions and specificities. They are usually named after their substrate, their regulatory molecules, or some aspect of a mutant phenotype. With regard to substrates, the protein kinases may be roughly divided into two groups; those that phosphorylate tyrosine residues (protein tyrosine kinases, PTK) and those that phosphorylate serine or threonine residues (serine/threonine kinases, STK). A few protein kinases have dual specificity and phosphorylate threonine and tyrosine residues. Almost all kinases contain a similar 250-300 amino acid catalytic domain. The N-terminal domain, which contains subdomains I-IV, generally folds into a two-lobed structure, which binds and orients the ATP (or GTP) donor molecule. The larger C terminal lobe, which contains subdomains VI A-XI, binds the protein substrate and carries out the transfer of the gamma phosphate from ATP to the hydroxyl group of a serine, threonine, or tyrosine residue. Subdomain V spans the two lobes.

The kinases may be categorized into families by the different amino acid sequences (generally between 5 and 100 residues) located on either side of, or inserted into loops of, the kinase domain. These added amino acid sequences allow the regulation of each kinase as it recognizes and interacts with its target protein. The primary structure of the kinase domains is conserved and can be further subdivided into 11 subdomains. Each of the 11 subdomains contains specific residues and motifs or patterns of amino acids that are characteristic of that subdomain and are highly conserved (Hardie, G. and Hanks, S. (1995) The Protein Kinase Facts Books, Vol I:7-20 Academic Press, San Diego, Calif.).

The second messenger dependent protein kinases primarily mediate the effects of second messengers such as cyclic AMP (cAMP), cyclic GMP, inositol triphosphate, phosphatidylinositol, 3,4,5-triphosphate, cyclic-ADPribose, arachidonic acid, diacylglycerol and calcium-calmodulin. The cyclic-AMP dependent protein kinases (PKA) are important members of the STK family. Cyclic-AMP is an intracellular mediator of hormone action in all prokaryotic and animal cells that have been studied. Such hormone-induced cellular responses include thyroid hormone secretion, cortisol secretion, progesterone secretion, glycogen breakdown, bone resorption, and regulation of heart rate and force of heart muscle contraction. PKA is found in all animal cells and is thought to account for the effects of cyclic-AMP in most of these cells. Altered PKA expression is implicated in a variety of disorders and diseases including cancer, thyroid isorders, diabetes, atherosclerosis, and cardiovascular disease (Isselbacher, K. J. et al. (1994) Harrison's Principles of Internal Medicine, McGraw-Hill, New York, N.Y., pp. 416-431, 1887).

Calcium-calmodulin (CaM) dependent protein kinases are also members of STK family. Calmodulin is a calcium receptor that mediates many calcium regulated processes by binding to target proteins in response to the binding of calcium. The principle target protein in these processes is CaM dependent protein kinases. CaM-kinases are involved in regulation of smooth muscle contraction (MLC kinase), glycogen breakdown (phosphorylase kinase), and neurotransmission (CaM kinase I and CaM kinase II). CaM kinase I phosphorylates a variety of substrates including the neurotransmitter related proteins synapsin I and II, the gene transcription regulator, CREB, and the cystic fibrosis conductance regulator protein, CFTR (Haribabu, B. et al. (1995) EMBO Journal 14:3679-86). CaM II kinase also phosphorylates synapsin at different sites, and controls the synthesis of catecholamines in the brain through phosphorylation and activation of tyrosine hydroxylase. Many of the CaM kinases are activated by phosphorylation in addition to binding to CaM. The kinase may autophosphorylate itself, or be phosphorylated by another kinase as part of a “kinase cascade”.

Another ligand-activated protein kinase is 5′-AMP-activated protein kinase (AMPK) (Gao, G. et al. (1996) J. Biol Chem. 15:8675-81). Mammalian AMPK is a regulator of fatty acid and sterol synthesis through phosphorylation of the enzymes acetyl-CoA carboxylase and hydroxymethylglutaryl-CoA reductase and mediates responses of these pathways to cellular stresses such as heat shock and depletion of glucose and ATP. AMPK is a heterotrimeric complex comprised of a catalytic alpha subunit and two non-catalytic beta and gamma subunits that are believed to regulate the activity of the alpha subunit. Subunits of AMPK have a much wider distribution in non-lipogenic tissues such as brain, heart, spleen, and lung than expected. This distribution suggests that its role may extend beyond regulation of lipid metabolism alone.

The mitogen-activated protein kinases (MAP) are also members of the STK family. MAP kinases also regulate intracellular signaling pathways. They mediate signal transduction from the cell surface to the nucleus via phosphorylation cascades. Several subgroups have been identified, and each manifests different substrate specificities and responds to distinct extracellular stimuli (Egan, S. E. and Weinberg, R. A. (1993) Nature 365:781-783). MAP kinase signaling pathways are present in mammalian cells as well as in yeast. The extracellular stimuli that activate mammalian pathways include epidermal growth factor (EGF), ultraviolet light, hyperosmolar medium, heat shock, endotoxic lipopolysaccharide (LPS), and pro-inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1).

PRK (proliferation-related kinase) is a serum/cytokine inducible STK that is involved in regulation of the cell cycle and cell proliferation in human megakaroytic cells (Li, B. et al. (1 996) J. Biol. Chem. 271:19402-8). PRK is related to the polo (derived from humans polo gene) family of STKs implicated in cell division. PRK is downregulated in lung tumor tissue and may be a proto-oncogene whose deregulated expression in normal tissue leads to oncogenic transformation. Altered MAP kinase expression is implicated in a variety of disease conditions including cancer, inflammation, immune disorders, and disorders affecting growth and development.

The cyclin-dependent protein kinases (CDKs) are another group of STKs that control the progression of cells through the cell cycle. Cyclins are small regulatory proteins that act by binding to and activating CDKs that then trigger various phases of the cell cycle by phosphorylating and activating selected proteins involved in the mitotic process. CDKs are unique in that they require multiple inputs to become activated. In addition to the binding of cyclin, CDK activation requires the phosphorylation of a specific threonine residue and the dephosphorylation of a specific tyrosine residue.

Protein tyrosine kinases, PTKs, specifically phosphorylate tyrosine residues on their target proteins and may be divided into transmembrane, receptor PTKs and nontransmembrane, non-receptor PTKs. Transmembrane protein-tyrosine kinases are receptors for most growth factors. Binding of growth factor to the receptor activates the transfer of a phosphate group from ATP to selected tyrosine side chains of the receptor and other specific proteins. Growth factors (GF) associated with receptor PTKs include; epidermal GF, platelet-derived GF, fibroblast GF, hepatocyte GF, insulin and insulin-like GFs, nerve GF, vascular endothelial GF, and macrophage colony stimulating factor.

Non-receptor PTKs lack transmembrane regions and, instead, form complexes with the intracellular regions of cell surface receptors. Such receptors that function through non-receptor PTKs include those for cytokines, hormones (growth hormone and prolactin) and antigen-specific receptors on T and B lymphocytes.

Many of these PTKs were first identified as the products of mutant oncogenes in cancer cells where their activation was no longer subject to normal cellular controls. In fact, about one third of the known oncogenes encode PTKs, and it is well known that cellular transformation (oncogenesis) is often accompanied by increased tyrosine phosphorylation activity (Carbonneau H and Tonks N K (1992) Annu. Rev. Cell. Biol. 8:463-93). Regulation of PTK activity may therefore be an important strategy in controlling some types of cancer.

Membrane-associated guanylate kinase (MAGUK) proteins participate in the assembly of multiprotein complexes on the inner surface of the plasma membrane at regions of cell-cell contact. MAGUK proteins share a common modular structure that consists of 1 or 3 PDZ domains, a SRC homology 3 (SH3) domain, and a C-terminal guanylate kinase (GuK) domain. Using the yeast 2-hybrid system to identify proteins that interact with the C terminus of KRasB, Dobrosotskaya et al. (1997) cloned mouse cDNAs encoding a protein which they called Magil for ‘membrane-associated guanylate kinase with an inverted arrangement of protein-protein interaction domains. The predicted 1,171-amino acid protein has an N-terminal GuK domain, followed by 2 WW domains and 5 PDZ domains. Alternative splicing generates at least 3 transcripts that encode isoforms with unique C termini. Northern blot analysis revealed multiple Magil transcripts in many tissues. By using cell fractionation and Western blot analyses, a 148-kD isoform was discovered. The protein, containing nuclear localization signals in its C terminus, is located predominantly in the nucleus and a 137-kD isoform was found primarily in membrane and cytoplasmic fractions. Dobrosotskaya et al. (1997) stated that the amino acid sequence of mouse Magil is 93% identical to that of the partial human WWP3 identified by Pirozzi et al. (1997).

MAGUK involves in cell junction organization, tumor suppression, and signalling. Their structure includes one or three copies of a DHR or PDZ domain (discs-large homologous region or PSD-95/SAP90, discs-large ZO-1 homologous domain), an SH3 domain, and a guanylate kinase domain. MAGUKs were classified into two subfamilies: Dlg-like with three DHR/PDZ domains and p55-like with a single HR/PDZ domain. There is now a new subfamily whose members have a novel domain structure: a calcium/calmodulin-dependent protein kinase domain in the N-terminus as well as the DHR/PDZ, SH3 and GUK domains in the C-terminus. These new MAGUKs may regulate transmembrane molecules that bind calcium, calmodulin, or nucleotides, camguk (cmg) is a Drosophila member of this novel MAGUK subfamily.

By screening a human brain cDNA library for WW domain-containing proteins, Pirozzi et al. (1997) identified a partial WWP3 cDNA. A longer BAIAP1 cDNA with CAG trinucleotide repeats was isolated from a human brain cDNA library by Margolis et al. (1997). For a review of membrane-associated guanylate kinase, see Dobrosotskaya et al.,. J. Biol. Chem. 272: 31589-31597, 1997, Margolis et al., Hum. Genet. 100: 114-122, 1997, Pirozzi et al., J. Biol. Chem. 272: 14611-14616, 1997 and Dimitratos et al., Mech Dev. 63(1):127-30, 1997.

Kinase proteins, particularly members of the membrane-associated guanylate kinase subfamily, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown members of this subfamily of kinase proteins. The present invention advances the state of the art by providing previously unidentified human kinase proteins that have homology to members of the membrane-associated guanylate kinase subfamily.

SUMMARY OF THE INVENTION

The present invention is based in part on the identification of amino acid sequences of human kinase peptides and proteins that are related to the membrane-associated guanylate kinase subfamily, as well as allelic variants and other mammalian orthologs thereof. These unique peptide sequences, and nucleic acid sequences that encode these peptides, can be used as models for the development of human therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of human therapeutic agents that modulate kinase activity in cells and tissues that express the kinase. Experimental data as provided in FIG. 1 indicates expression in the placenta, thyroid gland, muscle rhabdomyosarcoma, adrenal gland, infant brain, breast, brain glioblastoma, brain glioblastoma, retina and human hippocampus. infant brain, breast, brain glioblastoma, brain glioblastoma, retina and human hippocampus.

DESCRIPTION OF THE FIGURE SHEETS

FIG. 1 provides the nucleotide sequence of a cDNA molecule or transcript sequence that encodes the kinase protein of the present invention. (SEQ ID NO:1) In addition, structure and functional information is provided, such as ATG start, stop and tissue distribution, where available, that allows one to readily determine specific uses of inventions based on this molecular sequence. Experimental data as provided in FIG. 1 indicates expression in the placenta, thyroid gland, muscle rhabdomyosarcoma, adrenal gland, infant brain, breast, brain glioblastoma, brain glioblastoma, retina and human hippocampus.

FIG. 2 provides the predicted amino acid sequence of the kinase of the present invention. (SEQ ID NO:2) In addition structure and functional information such as protein family, function, and modification sites is provided where available, allowing one to readily determine specific uses of inventions based on this molecular sequence.

FIG. 3 provides genomic sequences that span the gene encoding the kinase protein of the present invention. (SEQ ID NO:3) In addition structure and functional information, such as intron/exon structure, promoter location, etc., is provided where available, allowing one to readily determine specific uses of inventions based on this molecular sequence. FIG. 3 provides information on SNPs that have been found in the gene encoding the kinase protein of the present invention. SNPs were identified at 65 different nucleotide positions in introns and regions 5′ and 3′ of the ORF. Such SNPs in introns and outside the ORF may affect control/regulatory elements.

DETAILED DESCRIPTION OF THE INVENTION

General Description

The present invention is based on the sequencing of the human genome. During the sequencing and assembly of the human genome, analysis of the sequence information revealed previously unidentified fragments of the human genome that encode peptides that share structural and/or sequence homology to protein/peptide/domains identified and characterized within the art as being a kinase protein or part of a kinase protein and are related to the membrane-associated guanylate kinase subfamily. Utilizing these sequences, additional genomic sequences were assembled and transcript and/or cDNA sequences were isolated and characterized. Based on this analysis, the present invention provides amino acid sequences of human kinase peptides and proteins that are related to the membrane-associated guanylate kinase subfamily, nucleic acid sequences in the form of transcript sequences, cDNA sequences and/or genomic sequences that encode these kinase peptides and proteins, nucleic acid variation (allelic information), tissue distribution of expression, and information about the closest art known protein/peptide/domain that has structural or sequence homology to the kinase of the present invention.

In addition to being previously unknown, the peptides that are provided in the present invention are selected based on their ability to be used for the development of commercially important products and services. Specifically, the present peptides are selected based on homology and/or structural relatedness to known kinase proteins of the membrane-associated guanylate kinase subfamily and the expression pattern observed. Experimental data as provided in FIG. 1 indicates expression in the placenta, thyroid gland, muscle rhabdomyosarcoma, adrenal gland, infant brain, breast, brain glioblastoma, brain glioblastoma, retina and human hippocampus. The art has clearly established the commercial importance of members of this family of proteins and proteins that have expression patterns similar to that of the present gene. Some of the more specific features of the peptides of the present invention, and the uses thereof, are described herein, particularly in the Background of the Invention and in the annotation provided in the Figures, and/or are known within the art for each of the known membrane-associated guanylate kinase family or subfamily of kinase proteins.

Specific Embodiments

Peptide Molecules

The present invention provides nucleic acid sequences that encode protein molecules that have been identified as being members of the kinase family of proteins and are related to the membrane-associated guanylate kinase subfamily (protein sequences are provided in FIG. 2, transcript/cDNA sequences are provided in FIG. 1 and genomic sequences are provided in FIG. 3). The peptide sequences provided in FIG. 2, as well as the obvious variants described herein, particularly allelic variants as identified herein and using the information in FIG. 3, will be referred herein as the kinase peptides of the present invention, kinase peptides, or peptides/proteins of the present invention.

The present invention provides isolated peptide and protein molecules that consist of, consist essentially of, or comprise the amino acid sequences of the kinase peptides disclosed in the FIG. 2, (encoded by the nucleic acid molecule shown in FIG. 1, transcript/cDNA or FIG. 3, genomic sequence), as well as all obvious variants of these peptides that are within the art to make and use. Some of these variants are described in detail below.

As used herein, a peptide is said to be “isolated” or “purified” when it is substantially free of cellular material or free of chemical precursors or other chemicals. The peptides of the present invention can be purified to homogeneity or other degrees of purity. The level of purification will be based on the intended use. The critical feature is that the preparation allows for the desired function of the peptide, even if in the presence of considerable amounts of other components (the features of an isolated nucleic acid molecule is discussed below).

In some uses, “substantially free of cellular material” includes preparations of the peptide having less than about 30% (by dry weight) other proteins (i.e., contaminating protein), less than about 20% other proteins, less than about 10% other proteins, or less than about 5% other proteins. When the peptide is recombinantly produced, it can also be substantially free of culture medium, i.e., culture medium represents less than about 20% of the volume of the protein preparation.

The language “substantially free of chemical precursors or other chemicals” includes reparations of the peptide in which it is separated from chemical precursors or other chemicals that are involved in its synthesis. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of the kinase peptide having less than about 30% (by dry weight) chemical precursors or other chemicals, less than about 20% chemical precursors or other chemicals, less than about 10% chemical precursors or other chemicals, or less than about 5% chemical precursors or other chemicals.

The isolated kinase peptide can be purified from cells that naturally express it, purified from cells that have been altered to express it (recombinant), or synthesized using known protein synthesis methods. Experimental data as provided in FIG. 1 indicates expression in the placenta, thyroid gland, muscle rhabdomyosarcoma, adrenal gland, infant brain, breast, brain glioblastoma, brain glioblastoma, retina and human hippocampus. For example, a nucleic acid molecule encoding the kinase peptide is cloned into an expression vector, the expression vector introduced into a host cell and the protein expressed in the host cell. The protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Many of these techniques are described in detail below.

Accordingly, the present invention provides proteins that consist of the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). The amino acid sequence of such a protein is provided in FIG. 2. A protein consists of an amino acid sequence when the amino acid sequence is the final amino acid sequence of the protein.

The present invention further provides proteins that consist essentially of the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). A protein consists essentially of an amino acid sequence when such an amino acid sequence is present with only a few additional amino acid residues, for example from about 1 to about 100 or so additional residues, typically from 1 to about 20 additional residues in the final protein.

The present invention further provides proteins that comprise the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). A protein comprises an amino acid sequence when the amino acid sequence is at least part of the final amino acid sequence of the protein. In such a fashion, the protein can be only the peptide or have additional amino acid molecules, such as amino acid residues (contiguous encoded sequence) that are naturally associated with it or heterologous amino acid residues/peptide sequences. Such a protein can have a few additional amino acid residues or can comprise several hundred or more additional amino acids. The preferred classes of proteins that are comprised of the kinase peptides of the present invention are the naturally occurring mature proteins. A brief description of how various types of these proteins can be made/isolated is provided below.

The kinase peptides of the present invention can be attached to heterologous sequences to form chimeric or fusion proteins. Such chimeric and fusion proteins comprise a kinase peptide operatively linked to a heterologous protein having an amino acid sequence not substantially homologous to the kinase peptide. “Operatively linked” indicates that the kinase peptide and the heterologous protein are fused in-frame. The heterologous protein can be fused to the N-terminus or C-terminus of the kinase peptide.

In some uses, the fusion protein does not affect the activity of the kinase peptide per se. For example, the fusion protein can include, but is not limited to, enzymatic fusion proteins, for example beta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins, particularly poly-His fusions, can facilitate the purification of recombinant kinase peptide. In certain host cells (e.g., mammalian host cells), expression and/or secretion of a protein can be increased by using a heterologous signal sequence.

A chimeric or fusion protein can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different protein sequences are ligated together in-frame in accordance with conventional techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see Ausubel et al., Current Protocols in Molecular Biology, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST protein). A kinase peptide-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the kinase peptide.

As mentioned above, the present invention also provides and enables obvious variants of the amino acid sequence of the proteins of the present invention, such as naturally occurring mature forms of the peptide, allelic/sequence variants of the peptides, non-naturally occurring recombinantly derived variants of the peptides, and orthologs and paralogs of the peptides. Such variants can readily be generated using art-known techniques in the fields of recombinant nucleic acid technology and protein biochemistry. It is understood, however, that variants exclude any amino acid sequences disclosed prior to the invention.

Such variants can readily be identified/made using molecular techniques and the sequence information disclosed herein. Further, such variants can readily be distinguished from other peptides based on sequence and/or structural homology to the kinase peptides of the present invention. The degree of homology/identity present will be based primarily on whether the peptide is a functional variant or non-functional variant, the amount of divergence present in the paralog family and the evolutionary distance between the orthologs.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the length of a reference sequence is aligned for comparison purposes. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity and similarity between two sequences can be accomplished using a mathematical algorithm. (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (Devereux, J., et al., Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com), using aNWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against sequence databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol. 215:403-10 (1990)). BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the proteins of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (Nucleic Acids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

Full-length pre-processed forms, as well as mature processed forms, of proteins that comprise one of the peptides of the present invention can readily be identified as having complete sequence identity to one of the kinase peptides of the present invention as well as being encoded by the same genetic locus as the kinase peptide provided herein. As indicated by the data presented in FIG. 3, the map position was determined to be on chromosome 17 by ePCR.

Allelic variants of a kinase peptide can readily be identified as being a human protein having a high degree (significant) of sequence homology/identity to at least a portion of the kinase peptide as well as being encoded by the same genetic locus as the kinase peptide provided herein. Genetic locus can readily be determined based on the genornic information provided in FIG. 3, such as the genomic sequence mapped to the reference human. As indicated by the data presented in FIG. 3, the map position was determined to be on chromosome 17 by ePCR. As used herein, two proteins (or a region of the proteins) have significant homology when the amino acid sequences are typically at least about 70-80%, 80-90%, and more typically at least about 90-95% or more homologous. A significantly homologous amino acid sequence, according to the present invention, will be encoded by a nucleic acid sequence that will hybridize to a kinase peptide encoding nucleic acid molecule under stringent conditions as more fully described below.

FIG. 3 provides information on SNPs that have been identified in a gene encoding the kinase protein of the present invention. 65 SNP variants were found, including 2 indels (indicated by a “−”) and 4 SNPs in exons, of which 3 of these cause changes in the amino acid sequence (i.e., nonsynonymous SNPs). The changes in the amino acid sequence that these SNPs cause is indicated in FIG. 3 and can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference.

Paralogs of a kinase peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the kinase peptide, as being encoded by a gene from humans, and as having similar activity or function. Two proteins will typically be considered paralogs when the amino acid sequences are typically at least about 60% or greater, and more typically at least about 70% or greater homology through a given region or domain. Such paralogs will be encoded by a nucleic acid sequence that will hybridize to a kinase peptide encoding nucleic acid molecule under moderate to stringent conditions as more fully described below.

Orthologs of a kinase peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the kinase peptide as well as being encoded by a gene from another organism. Preferred orthologs will be isolated from mammals, preferably primates, for the development of human therapeutic targets and agents. Such orthologs will be encoded by a nucleic acid sequence that will hybridize to a kinase peptide encoding nucleic acid molecule under moderate to stringent conditions, as more fully described below, depending on the degree of relatedness of the two organisms yielding the proteins.

Non-naturally occurring variants of the kinase peptides of the present invention can readily be generated using recombinant techniques. Such variants include, but are not limited to deletions, additions and substitutions in the amino acid sequence of the kinase peptide. For example, one class of substitutions are conserved amino acid substitution. Such substitutions are those that substitute a given amino acid in a kinase peptide by another amino acid of like characteristics. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residues Ser and Thr; exchange of the acidic residues Asp and Glu; substitution between the amide residues Asn and Gln; exchange of the basic residues Lys and Arg; and replacements among the aromatic residues Phe and Tyr. Guidance concerning which amino acid changes are likely to be phenotypically silent are found in Bowie et al., Science 247:1306-1310 (1990).

Variant kinase peptides can be fully functional or can lack function in one or more activities, e.g. ability to bind substrate, ability to phosphorylate substrate, ability to mediate signaling, etc. Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions. FIG. 2 provides the result of protein analysis and can be used to identify critical domains/regions. Functional variants can also contain substitution of similar amino acids that result in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree.

Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region.

Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085 (1989)), particularly using the results provided in FIG. 2. The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as kinase activity or in assays such as an in vitro proliferative activity. Sites that are critical for binding partner/substrate binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al. Science 255:306-312 (1992)).

The present invention further provides fragments of the kinase peptides, in addition to proteins and peptides that comprise and consist of such fragments, particularly those comprising the residues identified in FIG. 2. The fragments to which the invention pertains, however, are not to be construed as encompassing fragments that may be disclosed publicly prior to the present invention.

As used herein, a fragment comprises at least 8, 10, 12, 14, 16, or more contiguous amino acid residues from a kinase peptide. Such fragments can be chosen based on the ability to retain one or more of the biological activities of the kinase peptide or could be chosen for the ability to perform a function, e.g. bind a substrate or act as an immunogen. Particularly important fragments are biologically active fragments, peptides that are, for example, about 8 or more amino acids in length. Such fragments will typically comprise a domain or motif of the kinase peptide, e.g., active site, a transmembrane domain or a substrate-binding domain. Further, possible fragments include, but are not limited to, domain or motif containing fragments, soluble peptide fragments, and fragments containing immunogenic structures. Predicted domains and functional sites are readily identifiable by computer programs well known and readily available to those of skill in the art (e.g., PROSITE analysis). The results of one such analysis are provided in FIG. 2.

Polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids. Further, many amino acids, including the terminal amino acids, may be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. Common modifications that occur naturally in kinase peptides are described in basic texts, detailed monographs, and the research literature, and they are well known to those of skill in the art (some of these features are identified in FIG. 2).

Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

Such modifications are well known to those of skill in the art and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, for instance, are described in most basic texts, such as Proteins—Structure and Molecular Properties, 2nd Ed., T.E. Creighton, W. H. Freeman and Company, New York (1993). Many detailed reviews are available on this subject, such as by Wold, F., Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol 182: 626-646 (1990)) and Rattan et al. (Ann. N. Y. Acad. Sci. 663:48-62 (1992)).

Accordingly, the kinase peptides of the present invention also encompass derivatives or analogs in which a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included, in which the mature kinase peptide is fused with another compound, such as a compound to increase the half-life of the kinase peptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature kinase peptide, such as a leader or secretory sequence or a sequence for purification of the mature kinase peptide or a pro-protein sequence.

Protein/Peptide Uses

The proteins of the present invention can be used in substantial and specific assays related to the functional information provided in the Figures; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its binding partner or ligand) in biological fluids; and as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state). Where the protein binds or potentially binds to another protein or ligand (such as, for example, in a kinase-effector protein interaction or kinase-ligand interaction), the protein can be used to identify the binding partner/ligand so as to develop a system to identify inhibitors of the binding interaction. Any or all of these uses are capable of being developed into reagent grade or kit format for commercialization as commercial products.

Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and “Methods in Enzymology: Guide to Molecular Cloning Techniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

The potential uses of the peptides of the present invention are based primarily on the source of the protein as well as the class/action of the protein. For example, kinases isolated from humans and their human/mammalian orthologs serve as targets for identifying agents for use in mammalian therapeutic applications, e.g. a human drug, particularly in modulating a biological or pathological response in a cell or tissue that expresses the kinase. Experimental data as provided in FIG. 1 indicates that kinase proteins of the present invention are expressed in the placenta, thyroid gland, muscle rhabdomyosarcoma, adrenal gland, infant brain, breast, brain glioblastoma, brain glioblastoma, retina by virtual northern blot. In addition, PCR-based tissue screening panel indicates expression in human hippocampus. A large percentage of pharmaceutical agents are being developed that modulate the activity of kinase proteins, particularly members of the membrane-associated guanylate kinase subfamily (see Background of the Invention). The structural and functional information provided in the Background and Figures provide specific and substantial uses for the molecules of the present invention, particularly in combination with the expression information provided in FIG. 1. Experimental data as provided in FIG. 1 indicates expression in the placenta, thyroid gland, muscle rhabdomyosarcoma, adrenal gland, infant brain, breast, brain glioblastoma, brain glioblastoma, retina and human hippocampus. Such uses can readily be determined using the information provided herein, that which is known in the art, and routine experimentation.

The proteins of the present invention (including variants and fragments that may have been disclosed prior to the present invention) are useful for biological assays related to kinases that are related to members of the membrane-associated guanylate kinase subfamily. Such assays involve any of the known kinase functions or activities or properties useful for diagnosis and treatment of kinase-related conditions that are specific for the subfamily of kinases that the one of the present invention belongs to, particularly in cells and tissues that express the kinase. Experimental data as provided in FIG. 1 indicates that kinase proteins of the present invention are expressed in the placenta, thyroid gland, muscle rhabdomyosarcoma, adrenal gland, infant brain, breast, brain glioblastoma, brain glioblastoma, retina by virtual northern blot. In addition, PCR-based tissue screening panel indicates expression in human hippocampus.

The proteins of the present invention are also useful in drug screening assays, in cell-based or cell-free systems. Cell-based systems can be native, i.e., cells that normally express the kinase, as a biopsy or expanded in cell culture. Experimental data as provided in FIG. 1 indicates expression in the placenta, thyroid gland, muscle rhabdomyosarcoma, adrenal gland, infant brain, breast, brain glioblastoma, brain glioblastoma, retina and human hippocampus. In an alternate embodiment, cell-based assays involve recombinant host cells expressing the kinase protein.

The polypeptides can be used to identify compounds that modulate kinase activity of the protein in its natural state or an altered form that causes a specific disease or pathology associated with the kinase. Both the kinases of the present invention and appropriate variants and fragments can be used in high-throughput screens to assay candidate compounds for the ability to bind to the kinase. These compounds can be further screened against a functional kinase to determine the effect of the compound on the kinase activity. Further, these compounds can be tested in animal or invertebrate systems to determine activity/effectiveness. Compounds can be identified that activate (agonist) or inactivate (antagonist) the kinase to a desired degree.

Further, the proteins of the present invention can be used to screen a compound for the ability to stimulate or inhibit interaction between the kinase protein and a molecule that normally interacts with the kinase protein, e.g. a substrate or a component of the signal pathway that the kinase protein normally interacts (for example, another kinase). Such assays typically include the steps of combining the kinase protein with a candidate compound under conditions that allow the kinase protein, or fragment, to interact with the target molecule, and to detect the formation of a complex between the protein and the target or to detect the biochemical consequence of the interaction with the kinase protein and the target, such as any of the associated effects of signal transduction such as protein phosphorylation, cAMP turnover, and adenylate cyclase activation, etc.

Candidate compounds include, for example, 1) peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam et al., Nature 354:82-84 (1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorial chemistry-derived molecular libraries made of D- and/or L-configuration amino acids; 2) phosphopeptides (e.g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang et al., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and single chain antibodies as well as Fab, F(ab′)₂, Fab expression library fragments, and epitope-binding fragments of antibodies); and 4) small organic and inorganic molecules (e.g., molecules obtained from combinatorial and natural product libraries).

One candidate compound is a soluble fragment of the receptor that competes for substrate binding. Other candidate compounds include mutant kinases or appropriate fragments containing mutations that affect kinase function and thus compete for substrate. Accordingly, a fragment that competes for substrate, for example with a higher affinity, or a fragment that binds substrate but does not allow release, is encompassed by the invention.

The invention further includes other end point assays to identify compounds that modulate (stimulate or inhibit) kinase activity. The assays typically involve an assay of events in the signal transduction pathway that indicate kinase activity. Thus, the phosphorylation of a substrate, activation of a protein, a change in the expression of genes that are up- or down-regulated in response to the kinase protein dependent signal cascade can be assayed.

Any of the biological or biochemical functions mediated by the kinase can be used as an endpoint assay. These include all of the biochemical or biochemical/biological events described herein, in the references cited herein, incorporated by reference for these endpoint assay targets, and other functions known to those of ordinary skill in the art or that can be readily identified using the information provided in the Figures, particularly FIG. 2. Specifically, a biological function of a cell or tissues that expresses the kinase can be assayed. Experimental data as provided in FIG. 1 indicates that kinase proteins of the present invention are expressed in the placenta, thyroid gland, muscle rhabdomyosarcoma, adrenal gland, infant brain, breast, brain glioblastoma, brain glioblastoma, retina by virtual northern blot. In addition, PCR-based tissue screening panel indicates expression in human hippocampus.

Binding and/or activating compounds can also be screened by using chimeric kinase proteins in which the amino terminal extracellular domain, or parts thereof, the entire transmembrane domain or subregions, such as any of the seven transmembrane segments or any of the intracellular or extracellular loops and the carboxy terminal intracellular domain, or parts thereof, can be replaced by heterologous domains or subregions. For example, a substrate-binding region can be used that interacts with a different substrate then that which is recognized by the native kinase. Accordingly, a different set of signal transduction components is available as an end-point assay for activation. This allows for assays to be performed in other than the specific host cell from which the kinase is derived.

The proteins of the present invention are also useful in competition binding assays in methods designed to discover compounds that interact with the kinase (e.g. binding partners and/or ligands). Thus, a compound is exposed to a kinase polypeptide under conditions that allow the compound to bind or to otherwise interact with the polypeptide. Soluble kinase polypeptide is also added to the mixture. If the test compound interacts with the soluble kinase polypeptide, it decreases the amount of complex formed or activity from the kinase target. This type of assay is particularly useful in cases in which compounds are sought that interact with specific regions of the kinase. Thus, the soluble polypeptide that competes with the target kinase region is designed to contain peptide sequences corresponding to the region of interest.

To perform cell free drug screening assays, it is sometimes desirable to immobilize either the kinase protein, or fragment, or its target molecule to facilitate separation of complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay.

Techniques for immobilizing proteins on matrices can be used in the drug screening assays. In one embodiment, a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the cell lysates (e.g., ³⁵S-labeled) and the candidate compound, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly, or in the supernatant after the complexes are dissociated. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of kinase-binding protein found in the bead fraction quantitated from the gel using standard electrophoretic techniques. For example, either the polypeptide or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin using techniques well known in the art. Alternatively, antibodies reactive with the protein but which do not interfere with binding of the protein to its target molecule can be derivatized to the wells of the plate, and the protein trapped in the wells by antibody conjugation. Preparations of a kinase-binding protein and a candidate compound are incubated in the kinase protein-presenting wells and the amount of complex trapped in the well can be quantitated. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the kinase protein target molecule, or which are reactive with kinase protein and compete with the target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the target molecule.

Agents that modulate one of the kinases of the present invention can be identified using one or more of the above assays, alone or in combination. It is generally preferable to use a cell-based or cell free system first and then confirm activity in an animal or other model system. Such model systems are well known in the art and can readily be employed in this context.

Modulators of kinase protein activity identified according to these drug screening assays can be used to treat a subject with a disorder mediated by the kinase pathway, by treating cells or tissues that express the kinase. Experimental data as provided in FIG. 1 indicates expression in the placenta, thyroid gland, muscle rhabdomyosarcoma, adrenal gland, infant brain, breast, brain glioblastoma, brain glioblastoma, retina and human hippocampus. These methods of treatment include the steps of administering a modulator of kinase activity in a pharmaceutical composition to a subject in need of such treatment, the modulator being identified as described herein.

In yet another aspect of the invention, the kinase proteins can be used as “bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO 94/10300), to identify other proteins, which bind to or interact with the kinase and are involved in kinase activity. Such kinase-binding proteins are also likely to be involved in the propagation of signals by the kinase proteins or kinase targets as, for example, downstream elements of a kinase-mediated signaling pathway. Alternatively, such kinase-binding proteins are likely to be kinase inhibitors.

The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a kinase protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. If the “bait” and the “prey” proteins are able to interact, in vivo, forming a kinase-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the kinase protein.

This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a kinase-modulating agent, an antisense kinase nucleic acid molecule, a kinase-specific antibody, or a kinase-binding partner) can be used in an animal or other model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal or other model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.

The kinase proteins of the present invention are also useful to provide a target for diagnosing a disease or predisposition to disease mediated by the peptide. Accordingly, the invention provides methods for detecting the presence, or levels of, the protein (or encoding mRNA) in a cell, tissue, or organism. Experimental data as provided in FIG. 1 indicates expression in the placenta, thyroid gland, muscle rhabdomyosarcoma, adrenal gland, infant brain, breast, brain glioblastoma, brain glioblastoma, retina and human hippocampus. The method involves contacting a biological sample with a compound capable of interacting with the kinase protein such that the interaction can be detected. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.

One agent for detecting a protein in a sample is an antibody capable of selectively binding to protein. A biological sample includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.

The peptides of the present invention also provide targets for diagnosing active protein activity, disease, or predisposition to disease, in a patient having a variant peptide, particularly activities and conditions that are known for other members of the family of proteins to which the present one belongs. Thus, the peptide can be isolated from a biological sample and assayed for the presence of a genetic mutation that results in aberrant peptide. This includes amino acid substitution, deletion, insertion, rearrangement, (as the result of aberrant splicing events), and inappropriate post-translational modification. Analytic methods include altered electrophoretic mobility, altered tryptic peptide digest, altered kinase activity in cell-based or cell-free assay, alteration in substrate or antibody-binding pattern, altered isoelectric point, direct amino acid sequencing, and any other of the known assay techniques useful for detecting mutations in a protein. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.

In vitro techniques for detection of peptide include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence using a detection reagent, such as an antibody or protein binding agent. Alternatively, the peptide can be detected in vivo in a subject by introducing into the subject a labeled anti-peptide antibody or other types of detection agent. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. Particularly usefull are methods that detect the allelic variant of a peptide expressed in a subject and methods which detect fragments of a peptide in a sample.

The peptides are also useful in pharmacogenomic analysis. Pharmacogenomics deal with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin. Chem. 43(2):254-266 (1997)). The clinical outcomes of these variations result in severe toxicity of therapeutic drugs in certain individuals or therapeutic failure of drugs in certain individuals as a result of individual variation in metabolism. Thus, the genotype of the individual can determine the way a therapeutic compound acts on the body or the way the body metabolizes the compound. Further, the activity of drug metabolizing enzymes effects both the intensity and duration of drug action. Thus, the pharmacogenomics of the individual permit the selection of effective compounds and effective dosages of such compounds for prophylactic or therapeutic treatment based on the individual's genotype. The discovery of genetic polymorphisms in some drug metabolizing enzymes has explained why some patients do not obtain the expected drug effects, show an exaggerated drug effect, or experience serious toxicity from standard drug dosages. Polymorphisms can be expressed in the phenotype of the extensive metabolizer and the phenotype of the poor metabolizer. Accordingly, genetic polymorphism may lead to allelic protein variants of the kinase protein in which one or more of the kinase functions in one population is different from those in another population. The peptides thus allow a target to ascertain a genetic predisposition that can affect treatment modality. Thus, in a ligand-based treatment, polymorphism may give rise to amino terminal extracellular domains and/or other substrate-binding regions that are more or less active in substrate binding, and kinase activation. Accordingly, substrate dosage would necessarily be modified to maximize the therapeutic effect within a given population containing a polymorphism. As an alternative to genotyping, specific polymorphic peptides could be identified.

The peptides are also useful for treating a disorder characterized by an absence of, inappropriate, or unwanted expression of the protein. Experimental data as provided in FIG. 1 indicates expression in the placenta, thyroid gland, muscle rhabdomyosarcoma, adrenal gland, infant brain, breast, brain glioblastoma, brain glioblastoma, retina and human hippocampus. Accordingly, methods for treatment include the use of the kinase protein or fragments.

Antibodies

The invention also provides antibodies that selectively bind to one of the peptides of the present invention, a protein comprising such a peptide, as well as variants and fragments thereof. As used herein, an antibody selectively binds a target peptide when it binds the target peptide and does not significantly bind to unrelated proteins. An antibody is still considered to selectively bind a peptide even if it also binds to other proteins that are not substantially homologous with the target peptide so long as such proteins share homology with a fragment or domain of the peptide target of the antibody. In this case, it would be understood that antibody binding to the peptide is still selective despite some degree of cross-reactivity.

As used herein, an antibody is defined in terms consistent with that recognized within the art: they are multi-subunit proteins produced by a mammalian organism in response to an antigen challenge. The antibodies of the present invention include polyclonal antibodies and monoclonal antibodies, as well as fragments of such antibodies, including, but not limited to, Fab or F(ab′)₂, and Fv fragments.

Many methods are known for generating and/or identifying antibodies to a given target peptide. Several such methods are described by Harlow, Antibodies, Cold Spring Harbor Press, (1989).

In general, to generate antibodies, an isolated peptide is used as an immunogen and is administered to a mammalian organism, such as a rat, rabbit or mouse. The full-length protein, an antigenic peptide fragment or a fusion protein can be used. Particularly important fragments are those covering functional domains, such as the domains identified in FIG. 2, and domain of sequence homology or divergence amongst the family, such as those that can readily be identified using protein alignment methods and as presented in the Figures.

Antibodies are preferably prepared from regions or discrete fragments of the kinase proteins. Antibodies can be prepared from any region of the peptide as described herein. However, preferred regions will include those involved in function/activity and/or kinase/binding partner interaction. FIG. 2 can be used to identify particularly important regions while sequence alignment can be used to identify conserved and unique sequence fragments.

An antigenic fragment will typically comprise at least 8 contiguous amino acid residues. The antigenic peptide can comprise, however, at least 10, 12, 14, 16 or more amino acid residues. Such fragments can be selected on a physical property, such as fragments correspond to regions that are located on the surface of the protein, e.g., hydrophilic regions or can be selected based on sequence uniqueness (see FIG. 2).

Detection on an antibody of the present invention can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

Antibody Uses

The antibodies can be used to isolate one of the proteins of the present invention by standard techniques, such as affinity chromatography or immunoprecipitation. The antibodies can facilitate the purification of the natural protein from cells and recombinantly produced protein expressed in host cells. In addition, such antibodies are useful to detect the presence of one of the proteins of the present invention in cells or tissues to determine the pattern of expression of the protein among various tissues in an organism and over the course of normal development. Experimental data as provided in FIG. 1 indicates that kinase proteins of the present invention are expressed in the placenta, thyroid gland, muscle rhabdomyosarcoma, adrenal gland, infant brain, breast, brain glioblastoma, brain glioblastoma, retina by virtual northern blot. In addition, PCR-based tissue screening panel indicates expression in human hippocampus. Further, such antibodies can be used to detect protein in situ, in vitro, or in a cell lysate or supernatant in order to evaluate the abundance and pattern of expression. Also, such antibodies can be used to assess abnormal tissue distribution or abnormal expression during development or progression of a biological condition. Antibody detection of circulating fragments of the full length protein can be used to identify turnover.

Further, the antibodies can be used to assess expression in disease states such as in active stages of the disease or in an individual with a predisposition toward disease related to the protein's function. When a disorder is caused by an inappropriate tissue distribution, developmental expression, level of expression of the protein, or expressed/processed form, the antibody can be prepared against the normal protein. Experimental data as provided in FIG. 1 indicates expression in the placenta, thyroid gland, muscle rhabdomyosarcoma, adrenal gland, infant brain, breast, brain glioblastoma, brain glioblastoma, retina and human hippocampus. If a disorder is characterized by a specific mutation in the protein, antibodies specific for this mutant protein can be used to assay for the presence of the specific mutant protein.

The antibodies can also be used to assess normal and aberrant subcellular localization of cells in the various tissues in an organism. Experimental data as provided in FIG. 1 indicates expression in the placenta, thyroid gland, muscle rhabdomyosarcoma, adrenal gland, infant brain, breast, brain glioblastoma, brain glioblastoma, retina and human hippocampus. The diagnostic uses can be applied, not only in genetic testing, but also in monitoring a treatment modality. Accordingly, where treatment is ultimately aimed at correcting expression level or the presence of aberrant sequence and aberrant tissue distribution or developmental expression, antibodies directed against the protein or relevant fragments can be used to monitor therapeutic efficacy.

Additionally, antibodies are useful in pharmacogenomic analysis. Thus, antibodies prepared against polymorphic proteins can be used to identify individuals that require modified treatment modalities. The antibodies are also useful as diagnostic tools as an immunological marker for aberrant protein analyzed by electrophoretic mobility, isoelectric point, tryptic peptide digest, and other physical assays known to those in the art.

The antibodies are also useful for tissue typing. Experimental data as provided in FIG. 1 indicates expression in the placenta, thyroid gland, muscle rhabdomyosarcoma, adrenal gland, infant brain, breast, brain glioblastoma, brain glioblastoma, retina and human hippocampus. Thus, where a specific protein has been correlated with expression in a specific tissue, antibodies that are specific for this protein can be used to identify a tissue type.

The antibodies are also useful for inhibiting protein function, for example, blocking the binding of the kinase peptide to a binding partner such as a substrate. These uses can also be applied in a therapeutic context in which treatment involves inhibiting the protein's function. An antibody can be used, for example, to block binding, thus modulating (agonizing or antagonizing) the peptides activity. Antibodies can be prepared against specific fragments containing sites required for function or against intact protein that is associated with a cell or cell membrane. See FIG. 2 for structural information relating to the proteins of the present invention.

The invention also encompasses kits for using antibodies to detect the presence of a protein in a biological sample. The kit can comprise antibodies such as a labeled or labelable antibody and a compound or agent for detecting protein in a biological sample; means for determining the amount of protein in the sample; means for comparing the amount of protein in the sample with a standard; and instructions for use. Such a kit can be supplied to detect a single protein or epitope or can be configured to detect one of a multitude of epitopes, such as in an antibody detection array. Arrays are described in detail below for nuleic acid arrays and similar methods have been developed for antibody arrays.

Nucleic Acid Molecules

The present invention further provides isolated nucleic acid molecules that encode a kinase peptide or protein of the present invention (cDNA, transcript and genomic sequence). Such nucleic acid molecules will consist of, consist essentially of, or comprise a nucleotide sequence that encodes one of the kinase peptides of the present invention, an allelic variant thereof, or an ortholog or paralog thereof.

As used herein, an “isolated” nucleic acid molecule is one that is separated from other nucleic acid present in the natural source of the nucleic acid. Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. However, there can be some flanking nucleotide sequences, for example up to about 5 KB, 4 KB, 3 KB, 2 KB, or 1 KB or less, particularly contiguous peptide encoding sequences and peptide encoding sequences within the same gene but separated by introns in the genomic sequence. The important point is that the nucleic acid is isolated from remote and unimportant flanking sequences such that it can be subjected to the specific manipulations described herein such as recombinant expression, preparation of probes and primers, and other uses specific to the nucleic acid sequences.

Moreover, an “isolated” nucleic acid molecule, such as a transcript/cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. However, the nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated.

For example, recombinant DNA molecules contained in a vector are considered isolated. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the isolated DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.

Accordingly, the present invention provides nucleic acid molecules that consist of the nucleotide sequence shown in FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule consists of a nucleotide sequence when the nucleotide sequence is the complete nucleotide sequence of the nucleic acid molecule.

The present invention further provides nucleic acid molecules that consist essentially of the nucleotide sequence shown in FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule consists essentially of a nucleotide sequence when such a nucleotide sequence is present with only a few additional nucleic acid residues in the final nucleic acid molecule.

The present invention further provides nucleic acid molecules that comprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule comprises a nucleotide sequence when the nucleotide sequence is at least part of the final nucleotide sequence of the nucleic acid molecule. In such a fashion, the nucleic acid molecule can be only the nucleotide sequence or have additional nucleic acid residues, such as nucleic acid residues that are naturally associated with it or heterologous nucleotide sequences. Such a nucleic acid molecule can have a few additional nucleotides or can comprises several hundred or more additional nucleotides. A brief description of how various types of these nucleic acid molecules can be readily made/isolated is provided below.

In FIGS. 1 and 3, both coding and non-coding sequences are provided. Because of the source of the present invention, humans genomic sequence (FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleic acid molecules in the Figures will contain genomic intronic sequences, 5′ and 3′ non-coding sequences, gene regulatory regions and non-coding intergenic sequences. In general such sequence features are either noted in FIGS. 1 and 3 or can readily be identified using computational tools known in the art. As discussed below, some of the non-coding regions, particularly gene regulatory elements such as promoters, are useful for a variety of purposes, e.g. control of heterologous gene expression, target for identifying gene activity modulating compounds, and are particularly claimed as fragments of the genomic sequence provided herein.

The isolated nucleic acid molecules can encode the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature peptide (when the mature form has more than one peptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, facilitate protein trafficking, prolong or shorten protein half-life or facilitate manipulation of a protein for assay or production, among other things. As generally is the case in situ, the additional amino acids may be processed away from the mature protein by cellular enzymes.

As mentioned above, the isolated nucleic acid molecules include, but are not limited to, the sequence encoding the kinase peptide alone, the sequence encoding the mature peptide and additional coding sequences, such as a leader or secretory sequence (e.g., a pre-pro or pro-protein sequence), the sequence encoding the mature peptide, with or without the additional coding sequences, plus additional non-coding sequences, for example introns and non-coding 5′ and 3′ sequences such as transcribed but non-translated sequences that play a role in transcription, mRNA processing (including splicing and polyadenylation signals), ribosome binding and stability of mRNA. In addition, the nucleic acid molecule may be fused to a marker sequence encoding, for example, a peptide that facilitates purification.

Isolated nucleic acid molecules can be in the form of RNA, such as mRNA, or in the form DNA, including cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof The nucleic acid, especially DNA, can be double-stranded or single-stranded. Single-stranded nucleic acid can be the coding strand (sense strand) or the non-coding strand (anti-sense strand).

The invention further provides nucleic acid molecules that encode fragments of the peptides of the present invention as well as nucleic acid molecules that encode obvious variants of the kinase proteins of the present invention that are described above. Such nucleic acid molecules may be naturally occurring, such as allelic variants (same locus), paralogs (different locus), and orthologs (different organism), or may be constructed by recombinant DNA methods or by chemical synthesis. Such non-naturally occurring variants may be made by mutagenesis techniques, including those applied to nucleic acid molecules, cells, or organisms. Accordingly, as discussed above, the variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions.

The present invention further provides non-coding fragments of the nucleic acid molecules provided in FIGS. 1 and 3. Preferred non-coding fragments include, but are not limited to, promoter sequences, enhancer sequences, gene modulating sequences and gene termination sequences. Such fragments are useful in controlling heterologous gene expression and in developing screens to identify gene-modulating agents. A promoter can readily be identified as being 5′ to the ATG start site in the genomic sequence provided in FIG. 3.

A fragment comprises a contiguous nucleotide sequence greater than 12 or more nucleotides. Further, a fragment could at least 30, 40, 50, 100, 250 or 500 nucleotides in length. The length of the fragment will be based on its intended use. For example, the fragment can encode epitope bearing regions of the peptide, or can be useful as DNA probes and primers. Such fragments can be isolated using the known nucleotide sequence to synthesize an oligonucleotide probe. A labeled probe can then be used to screen a cDNA library, genomic DNA library, or mRNA to isolate nucleic acid corresponding to the coding region. Further, primers can be used in PCR reactions to clone specific regions of gene.

A probe/primer typically comprises substantially a purified oligonucleotide or oligonucleotide pair. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 20, 25, 40, 50 or more consecutive nucleotides.

Orthologs, homologs, and allelic variants can be identified using methods well known in the art. As described in the Peptide Section, these variants comprise a nucleotide sequence encoding a peptide that is typically 60-70%, 70-80%, 80-90%, and more typically at least about 90-95% or more homologous to the nucleotide sequence shown in the Figure sheets or a fragment of this sequence. Such nucleic acid molecules can readily be identified as being able to hybridize under moderate to stringent conditions, to the nucleotide sequence shown in the Figure sheets or a fragment of the sequence. Allelic variants can readily be determined by genetic locus of the encoding gene. As indicated by the data presented in FIG. 3, the map position was determined to be on chromosome 17 by ePCR.

FIG. 3 provides information on SNPs that have been identified in a gene encoding the kinase protein of the present invention. 65 SNP variants were found, including 2 indels (indicated by a “−”) and 4 SNPs in exons, of which 3 of these cause changes in the amino acid sequence (i.e., nonsynonymous SNPs). The changes in the amino acid sequence that these SNPs cause is indicated in FIG. 3 and can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference.

As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences encoding a peptide at least 60-70% homologous to each other typically remain hybridized to each other. The conditions can be such that sequences at least about 60%, at least about 70%, or at least about 80% or more homologous to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example of stringent hybridization conditions are hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45 C, followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65 C. Examples of moderate to low stringency hybridization conditions are well known in the art.

Nucleic Acid Molecule Uses

The nucleic acid molecules of the present invention are useful for probes, primers, chemical intermediates, and in biological assays. The nucleic acid molecules are useful as a hybridization probe for messenger RNA, transcript/cDNA and genomic DNA to isolate full-length cDNA and genomic clones encoding the peptide described in FIG. 2 and to isolate cDNA and genomic clones that correspond to variants (alleles, orthologs, etc.) producing the same or related peptides shown in FIG. 2. FIG. 3 provides information on SNPs that have been found in the gene encoding the kinase protein of the present invention. SNPs were identified at 65 different nucleotide positions in introns and regions 5′ and 3′ of the ORF. Such SNPs in introns and outside the ORF may affect control/regulatory elements.

The probe can correspond to any sequence along the entire length of the nucleic acid molecules provided in the Figures. Accordingly, it could be derived from 5′ noncoding regions, the coding region, and 3′ noncoding regions. However, as discussed, fragments are not to be construed as encompassing fragments disclosed prior to the present invention.

The nucleic acid molecules are also useful as primers for PCR to amplify any given region of a nucleic acid molecule and are useful to synthesize antisense molecules of desired length and sequence.

The nucleic acid molecules are also useful for constructing recombinant vectors. Such vectors include expression vectors that express a portion of, or all of, the peptide sequences. Vectors also include insertion vectors, used to integrate into another nucleic acid molecule sequence, such as into the cellular genome, to alter in situ expression of a gene and/or gene product. For example, an endogenous coding sequence can be replaced via homologous recombination with all or part of the coding region containing one or more specifically introduced mutations.

The nucleic acid molecules are also useful for expressing antigenic portions of the proteins.

The nucleic acid molecules are also useful as probes for determining the chromosomal positions of the nucleic acid molecules by means of in situ hybridization methods. As indicated by the data presented in FIG. 3, the map position was determined to be on chromosome 17 by ePCR.

The nucleic acid molecules are also useful in making vectors containing the gene regulatory regions of the nucleic acid molecules of the present invention.

The nucleic acid molecules are also useful for designing ribozymes corresponding to all, or a part, of the mRNA produced from the nucleic acid molecules described herein.

The nucleic acid molecules are also useful for making vectors that express part, or all, of the peptides.

The nucleic acid molecules are also useful for constructing host cells expressing a part, or all, of the nucleic acid molecules and peptides.

The nucleic acid molecules are also useful for constructing transgenic animals expressing all, or a part, of the nucleic acid molecules and peptides.

The nucleic acid molecules are also useful as hybridization probes for determining the presence, level, form and distribution of nucleic acid expression. Experimental data as provided in FIG. 1 indicates that kinase proteins of the present invention are expressed in the placenta, thyroid gland, muscle rhabdomyosarcoma, adrenal gland, infant brain, breast, brain glioblastoma, brain glioblastoma, retina by virtual northern blot. In addition, PCR-based tissue screening panel indicates expression in human hippocampus. Accordingly, the probes can be used to detect the presence of, or to determine levels of, a specific nucleic acid molecule in cells, tissues, and in organisms. The nucleic acid whose level is determined can be DNA or RNA. Accordingly, probes corresponding to the peptides described herein can be used to assess expression and/or gene copy number in a given cell, tissue, or organism. These uses are relevant for diagnosis of disorders involving an increase or decrease in kinase protein expression relative to normal results.

In vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detecting DNA includes Southern hybridizations and in situ hybridization.

Probes can be used as a part of a diagnostic test kit for identifying cells or tissues that express a kinase protein, such as by measuring a level of a kinase-encoding nucleic acid in a sample of cells from a subject e.g., mRNA or genomic DNA, or determining if a kinase gene has been mutated. Experimental data as provided in FIG. 1 indicates that kinase proteins of the present invention are expressed in the placenta, thyroid gland, muscle rhabdomyosarcoma, adrenal gland, infant brain, breast, brain glioblastoma, brain glioblastoma, retina by virtual northern blot. In addition, PCR-based tissue screening panel indicates expression in human hippocampus.

Nucleic acid expression assays are useful for drug screening to identify compounds that modulate kinase nucleic acid expression.

The invention thus provides a method for identifying a compound that can be used to treat a disorder associated with nucleic acid expression of the kinase gene, particularly biological and pathological processes that are mediated by the kinase in cells and tissues that express it. Experimental data as provided in FIG. 1 indicates expression in the placenta, thyroid gland, muscle rhabdomyosarcoma, adrenal gland, infant brain, breast, brain glioblastoma, brain glioblastoma, retina and human hippocampus. The method typically includes assaying the ability of the compound to modulate the expression of the kinase nucleic acid and thus identifying a compound that can be used to treat a disorder characterized by undesired kinase nucleic acid expression. The assays can be performed in cell-based and cell-free systems. Cell-based assays include cells naturally expressing the kinase nucleic acid or recombinant cells genetically engineered to express specific nucleic acid sequences.

The assay for kinase nucleic acid expression can involve direct assay of nucleic acid levels, such as mRNA levels, or on collateral compounds involved in the signal pathway. Further, the expression of genes that are up- or down-regulated in response to the kinase protein signal pathway can also be assayed. In this embodiment the regulatory regions of these genes can be operably linked to a reporter gene such as luciferase.

Thus, modulators of kinase gene expression can be identified in a method wherein a cell is contacted with a candidate compound and the expression of mRNA determined. The level of expression of kinase mRNA in the presence of the candidate compound is compared to the level of expression of kinase mRNA in the absence of the candidate compound. The candidate compound can then be identified as a modulator of nucleic acid expression based on this comparison and be used, for example to treat a disorder characterized by aberrant nucleic acid expression. When expression of mRNA is statistically significantly greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of nucleic acid expression. When nucleic acid expression is statistically significantly less in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of nucleic acid expression.

The invention further provides methods of treatment, with the nucleic acid as a target, using a compound identified through drug screening as a gene modulator to modulate kinase nucleic acid expression in cells and tissues that express the kinase. Experimental data as provided in FIG. 1 indicates that kinase proteins of the present invention are expressed in the placenta, thyroid gland, muscle rhabdomyosarcoma, adrenal gland, infant brain, breast, brain glioblastoma, brain glioblastoma, retina by virtual northern blot. In addition, PCR-based tissue screening panel indicates expression in human hippocampus. Modulation includes both up-regulation (i.e. activation or agonization) or down-regulation (suppression or antagonization) or nucleic acid expression.

Alternatively, a modulator for kinase nucleic acid expression can be a small molecule or drug identified using the screening assays described herein as long as the drug or small molecule inhibits the kinase nucleic acid expression in the cells and tissues that express the protein. Experimental data as provided in FIG. 1 indicates expression in the placenta, thyroid gland, muscle rhabdomyosarcoma, adrenal gland, infant brain, breast, brain glioblastoma, brain glioblastoma, retina and human hippocampus.

The nucleic acid molecules are also useful for monitoring the effectiveness of modulating compounds on the expression or activity of the kinase gene in clinical trials or in a treatment regimen. Thus, the gene expression pattern can serve as a barometer for the continuing effectiveness of treatment with the compound, particularly with compounds to which a patient can develop resistance. The gene expression pattern can also serve as a marker indicative of a physiological response of the affected cells to the compound. Accordingly, such monitoring would allow either increased administration of the compound or the administration of alternative compounds to which the patient has not become resistant. Similarly, if the level of nucleic acid expression falls below a desirable level, administration of the compound could be commensurately decreased.

The nucleic acid molecules are also useful in diagnostic assays for qualitative changes in kinase nucleic acid expression, and particularly in qualitative changes that lead to pathology. The nucleic acid molecules can be used to detect mutations in kinase genes and gene expression products such as mRNA. The nucleic acid molecules can be used as hybridization probes to detect naturally occurring genetic mutations in the kinase gene and thereby to determine whether a subject with the mutation is at risk for a disorder caused by the mutation. Mutations include deletion, addition, or substitution of one or more nucleotides in the gene, chromosomal rearrangement, such as inversion or transposition, modification of genomic DNA, such as aberrant methylation patterns or changes in gene copy number, such as amplification. Detection of a mutated form of the kinase gene associated with a dysfinction provides a diagnostic tool for an active disease or susceptibility to disease when the disease results from overexpression, underexpression, or altered expression of a kinase protein.

Individuals carrying mutations in the kinase gene can be detected at the nucleic acid level by a variety of techniques. FIG. 3 provides information on SNPs that have been identified in a gene encoding the kinase protein of the present invention. 65 SNP variants were found, including 2 indels (indicated by a “−”) and 4 SNPs in exons, of which 3 of these cause changes in the amino acid sequence (i.e., nonsynonymous SNPs). The changes in the amino acid sequence that these SNPs cause is indicated in FIG. 3 and can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference. As indicated by the data presented in FIG. 3, the map position was determined to be on chromosome 17 by ePCR. Genomic DNA can be analyzed directly or can be amplified by using PCR prior to analysis. RNA or cDNA can be used in the same way. In some uses, detection of the mutation involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al., Science 241:1077-1080 (1988); and Nakazawa et al., PNAS 91:360-364 (1994)), the latter of which can be particularly useful for detecting point mutations in the gene (see Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. Deletions and insertions can be detected by a change in size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to normal RNA or antisense DNA sequences.

Alternatively, mutations in a kinase gene can be directly identified, for example, by alterations in restriction enzyme digestion patterns determined by gel electrophoresis.

Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site. Perfectly matched sequences can be distinguished from mismatched sequences by nuclease cleavage digestion assays or by differences in melting temperature.

Sequence changes at specific locations can also be assessed by nuclease protection assays such as RNase and S1 protection or the chemical cleavage method. Furthermore, sequence differences between a mutant kinase gene and a wild-type gene can be determined by direct DNA sequencing. A variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Naeve, C. W., (1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv. Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem. Biotechnol. 38:147-159 (1993)).

Other methods for detecting mutations in the gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242 (1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth. Enzymol. 217:286-295 (1992)), electrophoretic mobility of mutant and wild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989); Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al., Genet. Anal. Tech. Appl. 9:73-79 (1992)), and movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (Myers et al., Nature 313:495 (1985)). Examples of other techniques for detecting point mutations include selective oligonucleotide hybridization, selective amplification, and selective primer extension.

The nucleic acid molecules are also useful for testing an individual for a genotype that while not necessarily causing the disease, nevertheless affects the treatment modality. Thus, the nucleic acid molecules can be used to study the relationship between an individual's genotype and the individual's response to a compound used for treatment (pharmacogenomic relationship). Accordingly, the nucleic acid molecules described herein can be used to assess the mutation content of the kinase gene in an individual in order to select an appropriate compound or dosage regimen for treatment. FIG. 3 provides information on SNPs that have been identified in a gene encoding the kinase protein of the present invention. 65 SNP variants were found, including 2 indels (indicated by a “−”) and 4 SNPs in exons, of which 3 of these cause changes in the amino acid sequence (i.e., nonsynonymous SNPs). The changes in the amino acid sequence that these SNPs cause is indicated in FIG. 3 and can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference.

Thus nucleic acid molecules displaying genetic variations that affect treatment provide a diagnostic target that can be used to tailor treatment in an individual. Accordingly, the production of recombinant cells and animals containing these polymorphisms allow effective clinical design of treatment compounds and dosage regimens.

The nucleic acid molecules are thus useful as antisense constructs to control kinase gene expression in cells, tissues, and organisms. A DNA antisense nucleic acid molecule is designed to be complementary to a region of the gene involved in transcription, preventing transcription and hence production of kinase protein. An antisense RNA or DNA nucleic acid molecule would hybridize to the mRNA and thus block translation of mRNA into kinase protein.

Alternatively, a class of antisense molecules can be used to inactivate mRNA in order to decrease expression of kinase nucleic acid. Accordingly, these molecules can treat a disorder characterized by abnormal or undesired kinase nucleic acid expression. This technique involves cleavage by means of ribozymes containing nucleotide sequences complementary to one or more regions in the mRNA that attenuate the ability of the mRNA to be translated. Possible regions include coding regions and particularly coding regions corresponding to the catalytic and other functional activities of the kinase protein, such as substrate binding.

The nucleic acid molecules also provide vectors for gene therapy in patients containing cells that are aberrant in kinase gene expression. Thus, recombinant cells, which include the patient's cells that have been engineered ex vivo and returned to the patient, are introduced into an individual where the cells produce the desired kinase protein to treat the individual.

The invention also encompasses kits for detecting the presence of a kinase nucleic acid in a biological sample. Experimental data as provided in FIG. 1 indicates that kinase proteins of the present invention are expressed in the placenta, thyroid gland, muscle rhabdomyosarcoma, adrenal gland, infant brain, breast, brain glioblastoma, brain glioblastoma, retina by virtual northern blot. In addition, PCR-based tissue screening panel indicates expression in human hippocampus. For example, the kit can comprise reagents such as a labeled or labelable nucleic acid or agent capable of detecting kinase nucleic acid in a biological sample; means for determining the amount of kinase nucleic acid in the sample; and means for comparing the amount of kinase nucleic acid in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect kinase protein mRNA or DNA.

Nucleic Acid Arrays

The present invention further provides nucleic acid detection kits, such as arrays or microarrays of nucleic acid molecules that are based on the sequence information provided in FIGS. 1 and 3 (SEQ ID NOS:1 and 3).

As used herein “Arrays” or “Microarrays” refers to an array of distinct polynucleotides or oligonucleotides synthesized on a substrate, such as paper, nylon or other type of membrane, filter, chip, glass slide, or any other suitable solid support. In one embodiment, the microarray is prepared and used according to the methods described in U.S. Pat. No. 5,837,832, Chee et al., PCT application WO 95/11995 (Chee et al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of which are incorporated herein in their entirety by reference. In other embodiments, such arrays are produced by the methods described by Brown et al., U.S. Pat. No. 5,807,522.

The microarray or detection kit is preferably composed of a large number of unique, single-stranded nucleic acid sequences, usually either synthetic antisense oligonucleotides or fragments of cDNAs, fixed to a solid support. The oligonucleotides are preferably about 6-60 nucleotides in length, more preferably 15-30 nucleotides in length, and most preferably about 20-25 nucleotides in length. For a certain type of microarray or detection kit, it may be preferable to use oligonucleotides that are only 7-20 nucleotides in length. The microarray or detection kit may contain oligonucleotides that cover the known 5′, or 3′, sequence, sequential oligonucleotides which cover the full length sequence; or unique oligonucleotides selected from particular areas along the length of the sequence. Polynucleotides used in the microarray or detection kit may be oligonucleotides that are specific to a gene or genes of interest.

In order to produce oligonucleotides to a known sequence for a microarray or detection kit, the gene(s) of interest (or an ORF identified from the contigs of the present invention) is typically examined using a computer algorithm which starts at the 5′ or at the 3′ end of the nucleotide sequence. Typical algorithms will then identify oligomers of defined length that are unique to the gene, have a GC content within a range suitable for hybridization, and lack predicted secondary structure that may interfere with hybridization. In certain situations it may be appropriate to use pairs of oligonucleotides on a microarray or detection kit. The “pairs” will be identical, except for one nucleotide that preferably is located in the center of the sequence. The second oligonucleotide in the pair (mismatched by one) serves as a control. The number of oligonucleotide pairs may range from two to one million. The oligomers are synthesized at designated areas on a substrate using a light-directed chemical process. The substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support.

In another aspect, an oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application WO 95/251116 (Baldeschweiler et al.) which is incorporated herein in its entirety by reference. In another aspect, a “gridded” array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures. An array, such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other number between two and one million which lends itself to the efficient use of commercially available instrumentation.

In order to conduct sample analysis using a microarray or detection kit, the RNA or DNA from a biological sample is made into hybridization probes. The mRNA is isolated, and cDNA is produced and used as a template to make antisense RNA (aRNA). The aRNA is amplified in the presence of fluorescent nucleotides, and labeled probes are incubated with the microarray or detection kit so that the probe sequences hybridize to complementary oligonucleotides of the microarray or detection kit. Incubation conditions are adjusted so that hybridization occurs with precise complementary matches or with various degrees of less complementarity. After removal of nonhybridized probes, a scanner is used to determine the levels and patterns of fluorescence. The scanned images are examined to determine degree of complementarity and the relative abundance of each oligonucleotide sequence on the microarray or detection kit. The biological samples may be obtained from any bodily fluids (such as blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations. A detection system may be used to measure the absence, presence, and amount of hybridization for all of the distinct sequences simultaneously. This data may be used for large-scale correlation studies on the sequences, expression patterns, mutations, variants, or polymorphisms among samples.

Using such arrays, the present invention provides methods to identify the expression of the kinase proteins/peptides of the present invention. In detail, such methods comprise incubating a test sample with one or more nucleic acid molecules and assaying for binding of the nucleic acid molecule with components within the test sample. Such assays will typically involve arrays comprising many genes, at least one of which is a gene of the present invention and or alleles of the kinase gene of the present invention. FIG. 3 provides information on SNPs that have been identified in a gene encoding the kinase protein of the present invention. 65 SNP variants were found, including 2 indels (indicated by a “−”) and 4 SNPs in exons, of which 3 of these cause changes in the amino acid sequence (i.e., nonsynonymous SNPs). The changes in the amino acid sequence that these SNPs cause is indicated in FIG. 3 and can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference.

Conditions for incubating a nucleic acid molecule with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid molecule used in the assay. One skilled in the art will recognize that any one of the commonly available hybridization, amplification or array assay formats can readily be adapted to employ the novel fragments of the Human genome disclosed herein. Examples of such assays can be found in Chard, T, An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

The test samples of the present invention include cells, protein or membrane extracts of cells. The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing nucleic acid extracts or of cells are well known in the art and can be readily be adapted in order to obtain a sample that is compatible with the system utilized.

In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention.

Specifically, the invention provides a compartmentalized kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the nucleic acid molecules that can bind to a fragment of the Human genome disclosed herein; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound nucleic acid.

In detail, a compartmentalized kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers, strips of plastic, glass or paper, or arraying material such as silica. Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample, a container which contains the nucleic acid probe, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and containers which contain the reagents used to detect the bound probe. One skilled in the art will readily recognize that the previously unidentified kinase gene of the present invention can be routinely identified using the sequence information disclosed herein can be readily incorporated into one of the established kit formats which are well known in the art, particularly expression arrays.

Vectors/Host Cells

The invention also provides vectors containing the nucleic acid molecules described herein. The term “vector” refers to a vehicle, preferably a nucleic acid molecule, which can transport the nucleic acid molecules. When the vector is a nucleic acid molecule, the nucleic acid molecules are covalently linked to the vector nucleic acid. With this aspect of the invention, the vector includes a plasmid, single or double stranded phage, a single or double stranded RNA or DNA viral vector, or artificial chromosome, such as a BAC, PAC, YAC, OR MAC.

A vector can be maintained in the host cell as an extrachromosomal element where it replicates and produces additional copies of the nucleic acid molecules. Alternatively, the vector may integrate into the host cell genome and produce additional copies of the nucleic acid molecules when the host cell replicates.

The invention provides vectors for the maintenance (cloning vectors) or vectors for expression (expression vectors) of the nucleic acid molecules. The vectors can function in prokaryotic or eukaryotic cells or in both (shuttle vectors).

Expression vectors contain cis-acting regulatory regions that are operably linked in the vector to the nucleic acid molecules such that transcription of the nucleic acid molecules is allowed in a host cell. The nucleic acid molecules can be introduced into the host cell with a separate nucleic acid molecule capable of affecting transcription. Thus, the second nucleic acid molecule may provide a trans-acting factor interacting with the cis-regulatory control region to allow transcription of the nucleic acid molecules from the vector. Alternatively, a trans-acting factor may be supplied by the host cell. Finally, a trans-acting factor can be produced from the vector itself. It is understood, however, that in some embodiments, transcription and/or translation of the nucleic acid molecules can occur in a cell-free system.

The regulatory sequence to which the nucleic acid molecules described herein can be operably linked include promoters for directing mRNA transcription. These include, but are not limited to, the left promoter from bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, the early and late promoters from SV40, the CMV immediate early promoter, the adenovirus early and late promoters, and retrovirus long-terminal repeats.

In addition to control regions that promote transcription, expression vectors may also include regions that modulate transcription, such as repressor binding sites and enhancers. Examples include the SV40 enhancer, the cytomegalovirus immediate early enhancer, polyoma enhancer, adenovirus enhancers, and retrovirus LTR enhancers.

In addition to containing sites for transcription initiation and control, expression vectors can also contain sequences necessary for transcription termination and, in the transcribed region a ribosome binding site for translation. Other regulatory control elements for expression include initiation and termination codons as well as polyadenylation signals. The person of ordinary skill in the art would be aware of the numerous regulatory sequences that are useful in expression vectors. Such regulatory sequences are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

A variety of expression vectors can be used to express a nucleic acid molecule. Such vectors include chromosomal, episomal, and virus-derived vectors, for example vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, including yeast artificial chromosomes, from viruses such as baculoviruses, papovaviruses such as SV40, Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses, and retroviruses. Vectors may also be derived from combinations of these sources such as those derived from plasmid and bacteriophage genetic elements, e.g. cosmids and phagemids. Appropriate cloning and expression vectors for prokaryotic and eukaryotic hosts are described in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

The regulatory sequence may provide constitutive expression in one or more host cells (i.e. tissue specific) or may provide for inducible expression in one or more cell types such as by temperature, nutrient additive, or exogenous factor such as a hormone or other ligand. A variety of vectors providing for constitutive and inducible expression in prokaryotic and eukaryotic hosts are well known to those of ordinary skill in the art.

The nucleic acid molecules can be inserted into the vector nucleic acid by well-known methodology. Generally, the DNA sequence that will ultimately be expressed is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction enzymes and then ligating the fragments together. Procedures for restriction enzyme digestion and ligation are well known to those of ordinary skill in the art.

The vector containing the appropriate nucleic acid molecule can be introduced into an appropriate host cell for propagation or expression using well-known techniques. Bacterial cells include, but are not limited to, E coli, Streptomyces, and Salmonella typhimurium. Eukaryotic cells include, but are not limited to, yeast, insect cells such as Drosophila, animal cells such as COS and CHO cells, and plant cells.

As described herein, it may be desirable to express the peptide as a fusion protein. Accordingly, the invention provides fusion vectors that allow for the production of the peptides. Fusion vectors can increase the expression of a recombinant protein, increase the solubility of the recombinant protein, and aid in the purification of the protein by acting for example as a ligand for affinity purification. A proteolytic cleavage site may be introduced at the junction of the fusion moiety so that the desired peptide can ultimately be separated from the fusion moiety. Proteolytic enzymes include, but are not limited to, factor Xa, thrombin, and enterokinase. Typical fusion expression vectors include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J. ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., Gene 69:301-315(1988)) and pET 11d (Studier et al, Gene Expression Technology: Methods in Enzymology 185:60-89 (1990)).

Recombinant protein expression can be maximized in host bacteria by providing a genetic background wherein the host cell has an impaired capacity to proteolytically cleave the recombinant protein. (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)119-128). Alternatively, the sequence of the nucleic acid molecule of interest can be altered to provide preferential codon usage for a specific host cell, for example E. coli. (Wada et al, Nucleic Acids Res. 20:2111-2118 (1992)).

The nucleic acid molecules can also be expressed by expression vectors that are operative in yeast. Examples of vectors for expression in yeast e.g., S. cerevisiae include pYepSec1(aldari, et al., EMBO J. 6:229-234 (1987)), pMFa (Kurjan et al, Cell 30:933-943(1982)), pJRY88 (Schultz et al., Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, San Diego, Calif.)

The nucleic acid molecules can also be expressed in insect cells using, for example, baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al., Mol. Cell Biol. 3:2156-2165 (1983)) and the pVL series (Lucklow et al, Virology 170:31-39 (1989)).

In certain embodiments of the invention, the nucleic acid molecules described herein are expressed in mammalian cells using mammalian expression vectors. Examples of mammalian expression vectors include pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman et al., EMBO J. 6:187-195 (1987)).

The expression vectors listed herein are provided by way of example only of the well-known vectors available to those of ordinary skill in the art that would be useful to express the nucleic acid molecules. The person of ordinary skill in the art would be aware of other vectors suitable for maintenance propagation or expression of the nucleic acid molecules described herein. These are found for example in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning. A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

The invention also encompasses vectors in which the nucleic acid sequences described herein are cloned into the vector in reverse orientation, but operably linked to a regulatory sequence that permits transcription of antisense RNA. Thus, an antisense transcript can be produced to all, or to a portion, of the nucleic acid molecule sequences described herein, including both coding and non-coding regions. Expression of this antisense RNA is subject to each of the parameters described above in relation to expression of the sense RNA (regulatory sequences, constitutive or inducible expression, tissue-specific expression).

The invention also relates to recombinant host cells containing the vectors described herein. Host cells therefore include prokaryotic cells, lower eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, and higher eukaryotic cells such as mammalian cells.

The recombinant host cells are prepared by introducing the vector constructs described herein into the cells by techniques readily available to the person of ordinary skill in the art. These include, but are not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, lipofection, and other techniques such as those found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Host cells can contain more than one vector. Thus, different nucleotide sequences can be introduced on different vectors of the same cell. Similarly, the nucleic acid molecules can be introduced either alone or with other nucleic acid molecules that are not related to the nucleic acid molecules such as those providing trans-acting factors for expression vectors. When more than one vector is introduced into a cell, the vectors can be introduced independently, co-introduced or joined to the nucleic acid molecule vector.

In the case of bacteriophage and viral vectors, these can be introduced into cells as packaged or encapsulated virus by standard procedures for infection and transduction. Viral vectors can be replication-competent or replication-defective. In the case in which viral replication is defective, replication will occur in host cells providing functions that complement the defects.

Vectors generally include selectable markers that enable the selection of the subpopulation of cells that contain the recombinant vector constructs. The marker can be contained in the same vector that contains the nucleic acid molecules described herein or may be on a separate vector. Markers include tetracycline or ampicillin-resistance genes for prokaryotic host cells and dihydrofolate reductase or neomycin resistance for eukaryotic host cells. However, any marker that provides selection for a phenotypic trait will be effective.

While the mature proteins can be produced in bacteria, yeast, mammalian cells, and other cells under the control of the appropriate regulatory sequences, cell- free transcription and translation systems can also be used to produce these proteins using RNA derived from the DNA constructs described herein.

Where secretion of the peptide is desired, which is difficult to achieve with multi-transmembrane domain containing proteins such as kinases, appropriate secretion signals are incorporated into the vector. The signal sequence can be endogenous to the peptides or heterologous to these peptides.

Where the peptide is not secreted into the medium, which is typically the case with kinases, the protein can be isolated from the host cell by standard disruption procedures, including freeze thaw, sonication, mechanical disruption, use of lysing agents and the like. The peptide can then be recovered and purified by well-known purification methods including ammonium sulfate precipitation, acid extraction, anion or cationic exchange chromatography, phosphocellulose chromatography, hydrophobic-interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography, or high performance liquid chromatography.

It is also understood that depending upon the host cell in recombinant production of the peptides described herein, the peptides can have various glycosylation patterns, depending upon the cell, or maybe non-glycosylated as when produced in bacteria. In addition, the peptides may include an initial modified methionine in some cases as a result of a host-mediated process.

Uses of Vectors and Host Cells

The recombinant host cells expressing the peptides described herein have a variety of uses. First, the cells are useful for producing a kinase protein or peptide that can be further purified to produce desired amounts of kinase protein or fragments. Thus, host cells containing expression vectors are useful for peptide production.

Host cells are also useful for conducting cell-based assays involving the kinase protein or kinase protein fragments, such as those described above as well as other formats known in the art. Thus, a recombinant host cell expressing a native kinase protein is useful for assaying compounds that stimulate or inhibit kinase protein function.

Host cells are also useful for identifying kinase protein mutants in which these functions are affected. If the mutants naturally occur and give rise to a pathology, host cells containing the mutations are useful to assay compounds that have a desired effect on the mutant kinase protein (for example, stimulating or inhibiting function) which may not be indicated by their effect on the native kinase protein.

Genetically engineered host cells can be further used to produce non-human transgenic animals. A transgenic animal is preferably a mammal, for example a rodent, such as a rat or mouse, in which one or more of the cells of the animal include a transgene. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal in one or more cell types or tissues of the transgenic animal. These animals are useful for studying the function of a kinase protein and identifying and evaluating modulators of kinase protein activity. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, and amphibians.

A transgenic animal can be produced by introducing nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. Any of the kinase protein nucleotide sequences can be introduced as a transgene into the genome of a non-human animal, such as a mouse.

Any of the regulatory or other sequences useful in expression vectors can form part of the transgenic sequence. This includes intronic sequences and polyadenylation signals, if not already included. A tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the kinase protein to particular cells.

Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of transgenic mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene can further be bred to other transgenic animals carrying other transgenes. A transgenic animal also includes animals in which the entire animal or tissues in the animal have been produced using the homologously recombinant host cells described herein.

In another embodiment, transgenic non-human animals can be produced which contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage P1. For a description of the cre/loxP recombinase system, see, e.g., Lakso et al. PNAS 89:6232-6236 (1992). Another example of a recombinase system is the FLP recombinase system of S. cerevisiae (O'Gorman et al. Science 251:1351-1355 (1991). If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein is required. Such animals can be provided through the construction of “double” transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. Nature 385:810-813 (1997) and PCT International Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter G_(o) phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyst and then transferred to pseudopregnant female foster animal. The offspring born of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.

Transgenic animals containing recombinant cells that express the peptides described herein are useful to conduct the assays described herein in an in vivo context. Accordingly, the various physiological factors that are present in vivo and that could effect substrate binding, kinase protein activation, and signal transduction, may not be evident from in vitro cell-free or cell-based assays. Accordingly, it is useful to provide non-human transgenic animals to assay in vivo kinase protein function, including substrate interaction, the effect of specific mutant kinase proteins on kinase protein function and substrate interaction, and the effect of chimeric kinase proteins. It is also possible to assess the effect of null mutations, that is, mutations that substantially or completely eliminate one or more kinase protein functions.

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described modes for carrying out the invention which are obvious to those skilled in the field of molecular biology or related fields are intended to be within the scope of the following claims.

8 1 3604 DNA Human 1 gtggtctgtg cggtgtctcc caaaccacta cctggctgcc cccgtaccag ctgctgggaa 60 agactacttc ctccgagttg tgccggcctc cgcgttctcc tcacctcccc tctcctccgg 120 acctccgccc cctcgcggag aggccttgcc gctttaagag ccgggctagc gattgacaag 180 caataaacgc tgagcgcccg gctgcgctgg agccgcccgg agctaggggc ttcccgcggc 240 gcaggagaga cgtttcagag cccttgcctc cttcaccatg ccggttgccg ccaccaactc 300 tgaaactgcc atgcagcaag tcctggacaa cttgggatcc ctccccagtg ccacgggggc 360 tgcagagctg gacctgatct tccttcgagg cattatggaa agtcccatag taagatccct 420 ggccaaggcc catgagaggc tggaggagac gaagctggag gccgtgagag acaacaacct 480 ggagctggtg caggagatcc tgcgggacct ggcgcagctg gctgagcaga gcagcacagc 540 cgccgagctg gcccacatcc tccaggagcc ccacttccag tccctcctgg agacgcacga 600 ctctgtggcc tcaaagacct atgagacacc accccccagc cctggcctgg accctacgtt 660 cagcaaccag cctgtacctc ccgatgctgt gcgcatggtg ggcatccgca agacagccgg 720 agaacatctg ggtgtaacgt tccgcgtgga gggcggcgag ctggtgatcg cgcgcattct 780 gcatgggggc atggtggctc agcaaggcct gctgcatgtg ggtgacatca tcaaggaggt 840 gaacgggcag ccagtgggca gtgacccccg cgcactgcag gagctcctgc gcaatgccag 900 tggcagtgtc atcctcaaga tcctgcccag ctaccaggag ccccatctgc cccgccaggt 960 atttgtgaaa tgtcactttg actatgaccc ggcccgagac agcctcatcc cctgcaagga 1020 agcaggcctg cgcttcaacg ccggggactt gctccagatc gtaaaccagg atgatgccaa 1080 ctggtggcag gcatgccatg tcgaaggggg cagtgctggg ctcattccca gccagctgct 1140 ggaggagaag cggaaagcat ttgtcaagag ggacctggag ctgacaccaa actcagggac 1200 cctatgcggc agcctttcag gaaagaaaaa gaagcgaatg atgtatttga ccaccaagaa 1260 tgcagagttt gaccgtcatg agctgctcat ttatgaggag gtggcccgca tgcccccgtt 1320 ccgccggaaa accctggtac tgattggggc tcagggcgtg ggacggcgca gcctgaagaa 1380 caagctcatc atgtgggatc cagatcgcta tggcaccacg gtgccctaca cctcccggcg 1440 gccgaaagac tcagagcggg aaggtcaggg ttacagcttt gtgtcccgtg gggagatgga 1500 ggctgacgtc cgtgctgggc gctacctgga gcatggcgaa tacgagggca acctgtatgg 1560 cacacgtatt gactccatcc ggggcgtggt cgctgctggg aaggtgtgcg tgctggatgt 1620 caacccccag gcggtgaagg tgctacgaac ggccgagttt gtcccttacg tggtgttcat 1680 cgaggcccca gacttcgaga ccctgcgggc catgaacagg gctgcgctgg agagtggaat 1740 atccaccaag cagctcacgg aggcggacct gagacggaca gtggaggaga gcagccgcat 1800 ccagcggggc tacgggcact actttgacct ctgcctggtc aatagcaacc tggagaggac 1860 cttccgcgag ctccagacag ccatggagaa gctacggaca gagccccagt gggtgcctgt 1920 cagctgggtg tactgagcct gttcacctgg tccttggctc actctgtgtt gaaacccaga 1980 acctgaatcc atccccctcc tgacctgtga ccccctgcca caatccttag cccccatatc 2040 tggctgtcct tgggtaacag ctcccagcag gccctaagtc tggcttcagc acagaggcgt 2100 gcactgccag ggaggtgggc attcatgggg taccttgtgc ccaggtgctg cccactcctg 2160 atgcccattg gtcaccagat atctctgagg gccaagctat gcccaggaat gtgtcagagt 2220 cacctccata atggtcagta cagagaagag aaaagctgct ttgggaccac atggtcagta 2280 ggcacactgc cccctgccac ccctccccag tcaccagttc tcctctggac tggccacacc 2340 caccccattc ctggactcct cccacctctc acccctgtgt cggaggaaca ggccttgggc 2400 tgtttccgtg tgaccagggg aatgtgtggc ccgctggcag ccaggcaggc ccgggtggtg 2460 gtgccagcct ggtgccatct tgaaggctgg aggagtcaga gtgagagcca gtggccacag 2520 ctgcagagca ctgcagctcc cagctccttt ggaaagggac agggtcgcag ggcagatgct 2580 gctcggtcct tccctcatcc acagcttctc actgccgaag tttctccaga tttctccaat 2640 gtgtcctgac aggtcagccc tgctccccac agggccaggc tggcaggggc cagtgggctc 2700 agcccaggta ggggcaggat ggagggctga gccctgtgac aacctgctgt taccaactga 2760 agagccccaa gctctccatg gcccacagca ggcacaggtc tgagctctat gtccttgacc 2820 ttggtccatt tggttttctg tctagccagg tccaggtagc ccacttgcat cagggctgct 2880 gggttggagg ggctaaggag gagtgcagag gggaccttgg gagcctgggc ttgaaggaca 2940 gttgccctcc aggaggttcc tcacacacaa ctccagaggc gccatttaca ctgtagtctg 3000 tacaacctgt ggttccacgt gcatgttcgg cacctgtctg tgcctctggc accaggttgt 3060 gtgtgtgtgc gtgtgcacgt gcgtgtgtgt gtgtgtgtgt caggtttagt ttggggagga 3120 agcaaagggt tttgttttgg aggtcactct ttggggcccc tttctggggg ttccccatca 3180 gccctcattt cttataatac cctgatccca gactccaaag ccctggtcct ttcctgatgt 3240 ctcctccctt gtcttattgt ccccctaccc taaatgcccc cctgccataa cttggggagg 3300 gcagttttgt aaaataggag actcccttta agaaagaatg ctgtcctaga tgtacttggg 3360 catctcatcc ttcattattc tctgcattcc ttccgggggg agcctgtcct cagaggggac 3420 aacctgtgac accctgagtc caaacccttg tgcctcccag ttcttccaag tgtctaacta 3480 gtcttcgctg cagcgtcagc caaagctggc ccctgaacca ctgtgtgccc atttcctagg 3540 gaaggggaag gagaataaac agaatattta ttacaaaaaa aaaaaaaaaa aaaaaaaaaa 3600 aaaa 3604 2 552 PRT Human 2 Met Pro Val Ala Ala Thr Asn Ser Glu Thr Ala Met Gln Gln Val Leu 1 5 10 15 Asp Asn Leu Gly Ser Leu Pro Ser Ala Thr Gly Ala Ala Glu Leu Asp 20 25 30 Leu Ile Phe Leu Arg Gly Ile Met Glu Ser Pro Ile Val Arg Ser Leu 35 40 45 Ala Lys Ala His Glu Arg Leu Glu Glu Thr Lys Leu Glu Ala Val Arg 50 55 60 Asp Asn Asn Leu Glu Leu Val Gln Glu Ile Leu Arg Asp Leu Ala Gln 65 70 75 80 Leu Ala Glu Gln Ser Ser Thr Ala Ala Glu Leu Ala His Ile Leu Gln 85 90 95 Glu Pro His Phe Gln Ser Leu Leu Glu Thr His Asp Ser Val Ala Ser 100 105 110 Lys Thr Tyr Glu Thr Pro Pro Pro Ser Pro Gly Leu Asp Pro Thr Phe 115 120 125 Ser Asn Gln Pro Val Pro Pro Asp Ala Val Arg Met Val Gly Ile Arg 130 135 140 Lys Thr Ala Gly Glu His Leu Gly Val Thr Phe Arg Val Glu Gly Gly 145 150 155 160 Glu Leu Val Ile Ala Arg Ile Leu His Gly Gly Met Val Ala Gln Gln 165 170 175 Gly Leu Leu His Val Gly Asp Ile Ile Lys Glu Val Asn Gly Gln Pro 180 185 190 Val Gly Ser Asp Pro Arg Ala Leu Gln Glu Leu Leu Arg Asn Ala Ser 195 200 205 Gly Ser Val Ile Leu Lys Ile Leu Pro Ser Tyr Gln Glu Pro His Leu 210 215 220 Pro Arg Gln Val Phe Val Lys Cys His Phe Asp Tyr Asp Pro Ala Arg 225 230 235 240 Asp Ser Leu Ile Pro Cys Lys Glu Ala Gly Leu Arg Phe Asn Ala Gly 245 250 255 Asp Leu Leu Gln Ile Val Asn Gln Asp Asp Ala Asn Trp Trp Gln Ala 260 265 270 Cys His Val Glu Gly Gly Ser Ala Gly Leu Ile Pro Ser Gln Leu Leu 275 280 285 Glu Glu Lys Arg Lys Ala Phe Val Lys Arg Asp Leu Glu Leu Thr Pro 290 295 300 Asn Ser Gly Thr Leu Cys Gly Ser Leu Ser Gly Lys Lys Lys Lys Arg 305 310 315 320 Met Met Tyr Leu Thr Thr Lys Asn Ala Glu Phe Asp Arg His Glu Leu 325 330 335 Leu Ile Tyr Glu Glu Val Ala Arg Met Pro Pro Phe Arg Arg Lys Thr 340 345 350 Leu Val Leu Ile Gly Ala Gln Gly Val Gly Arg Arg Ser Leu Lys Asn 355 360 365 Lys Leu Ile Met Trp Asp Pro Asp Arg Tyr Gly Thr Thr Val Pro Tyr 370 375 380 Thr Ser Arg Arg Pro Lys Asp Ser Glu Arg Glu Gly Gln Gly Tyr Ser 385 390 395 400 Phe Val Ser Arg Gly Glu Met Glu Ala Asp Val Arg Ala Gly Arg Tyr 405 410 415 Leu Glu His Gly Glu Tyr Glu Gly Asn Leu Tyr Gly Thr Arg Ile Asp 420 425 430 Ser Ile Arg Gly Val Val Ala Ala Gly Lys Val Cys Val Leu Asp Val 435 440 445 Asn Pro Gln Ala Val Lys Val Leu Arg Thr Ala Glu Phe Val Pro Tyr 450 455 460 Val Val Phe Ile Glu Ala Pro Asp Phe Glu Thr Leu Arg Ala Met Asn 465 470 475 480 Arg Ala Ala Leu Glu Ser Gly Ile Ser Thr Lys Gln Leu Thr Glu Ala 485 490 495 Asp Leu Arg Arg Thr Val Glu Glu Ser Ser Arg Ile Gln Arg Gly Tyr 500 505 510 Gly His Tyr Phe Asp Leu Cys Leu Val Asn Ser Asn Leu Glu Arg Thr 515 520 525 Phe Arg Glu Leu Gln Thr Ala Met Glu Lys Leu Arg Thr Glu Pro Gln 530 535 540 Trp Val Pro Val Ser Trp Val Tyr 545 550 3 32654 DNA Human 3 cccccgggcc agctcctatc ccaagacctc cgccttcttt tcgtcctgac acggcactta 60 ggaccctatt cactgggttt aaactccaca ggcccctttc acccaggccc gttccctccc 120 tgtccacccg cccccgcccc tgggccctct ttctggggtg ctggttggga gaaccaagcc 180 ccgctgccca ttgacactgg tccccgcccc tgcggtaggg gctccaaagt ggcttcaggc 240 accaaggtgg tgagctgtga ggggccccgg tatctgcgcg gcctccccgg gggctgtgcg 300 ggttgggggg acttagaagc ccggcattac gtaagaaccg tgctgttagc gcttccgggg 360 gttggggagc gagacagagg ttgcgctggg tcccggtggc cccctggtcc ccctaggcag 420 ctaacaccac aatcttccct gggagggggt ccgcagaagg gcgcgcgctg cttctcccgc 480 ttcccaagct gtagcggggc gggtcgggag ccagggttag ggtctggaga cttgggaggg 540 gaccggtgcc agaagcgaag agctagggtt agaaagttag gtcaattaag gggggggcgg 600 tgcggataaa gacggggtgg gggttggccg gggcagtcat ttagtgacct catccctggt 660 ctccggcggg cgcgcacgta gagagccttc tttgatggct cacaacacgc ccccctcccc 720 caggtcctag cgcccagggt gttagggctg ggtccgttgg ggcaggggtg atctgatggt 780 ccaagcatgc acgtccgcct ggcctttcga ccccatcctc tggccctcag ttctgtccag 840 acctccccgc gacctctcgc tctccaaagc gcctttgtca gacctttctt ctccgcacct 900 ctttgtatgt gtatggatgg agggatgggt gggaggttgg aagaaacctc tccctttcac 960 tgctggtccg ctcgcctgcg gacactatgg ctgatgtgga tatctcggag gcagagctga 1020 gagggcatct gggcccaggc ttttggaagc cgaggtgcag gttcccagat gtctgggtgc 1080 aggaccttgg cacggagcca ggctagcttg cctggcactg ccgttctcct gtggacacag 1140 ccaggccccg ccctctcaga ggcttggacg ggcgtggggg agcaaagcca gccgatgctc 1200 tgggaaccga ctccccggga gaaaacccag gcaggctggg caagacttcc gagcgcaggg 1260 atttcataca ggatcctccc tgccttttcc cgccctccac gctctaggga aggaggactt 1320 ctggagtccc ttctctgacc ctgtgaggga gagcagcgcc agccaggaga aggctccagg 1380 tcatcaaggc ctgggagggg gagggaaggg atccctcctc cctgctctct aggagtagag 1440 attggcacca ggggcacggg ccagcacagt ggatcctggg catttcttcc agcctgggtg 1500 tagcctgaat aggggtgccc ttcctctccc tgaaagcacc tttattccat gtcctcagcg 1560 gcagagctgg gaggaaaggg aaccatcccc ctcacttttg taatttctgt cacctgagct 1620 caggtgacca gctcaggcct gagaagggaa tgggagcaga gagggtttta ttgaaatcag 1680 gtactagtcc tctgggaaga atcctctttt ggggggaggt ggagttgccg tggttccccc 1740 acccccagga ctcagcaggc attcagtcct aaagtgggca ctgccagatg cttcccccca 1800 ctccccaccc ctgctgtgag ccactgcctc ctgttgtcat ggcaactggg aattagggtt 1860 ttatccagca gcacatgccc aggaaggcaa agggaagaag ggtggggggt aaccccctgc 1920 cctctcctgg gagaacgagg gagccactcc tagcctgggg aggccctgga atggaaactg 1980 gggctgtaga ctccatgctt ggttctgccc agagcacttg gtctccttgt gccagctctg 2040 agctaagaaa gggagagagg agagagagcc agccagaggg gccgtggaga ggtagatatc 2100 actctgtcag ccccccagat aaatgtggaa catcggcgct ttggtaggcc taggttccag 2160 aagcaaataa aatcaatgtg gttctgactc tcagggaacc tgcagtctag gagtactcat 2220 gttttctact ttggctcaaa ttctggactc ttttattatt ttttaaatga cttttttttt 2280 tttaagacag gttcttgctc tgttgcccag gctggagtgc agtggcgcaa tcatggctta 2340 ccgtagcctc gacctcctgg gctcaagaga tccccctgcc tcagccccac aagtagctgg 2400 gatcataggc atgcaccacc acacctggat aatttttaat ttttttgtag agatgggggt 2460 ctcgccatgt tgcccaggtg gctctcaaac tcctgggctc aggtgattct cctgcctcag 2520 cctcctaaag tgctgggatt ataggcgtga gccaccttgc tcagccaaat tatagtctta 2580 ccctctaaaa attctacttg tttcctcttc aaaaaacaaa caaacaaaaa atacaacagg 2640 aagaaacact ttatttatat acttattttt gagtcataaa acctggattt tcattttagc 2700 tctccttatt agctttgtga acttgggcaa atcagttaac ctcccttatc ctccctggtc 2760 tcctcctcct cagagaaata ggcgagtaat acctgctctt cctgcttccc aggaccgttg 2820 gaatcattgt tgttctccaa cccccacttt ctgggcagca ccttctcccc tacctttctg 2880 gctcgtcctg aaggggaggc tcttcctatt cccttcccca ttgctttgcc cttgtatctg 2940 ctcatcctcc tctctcccct ctccaggaga gacgtttcag agcccttgcc tccttcacca 3000 tgccggttgc cgccaccaac tctgaaactg gtaagaaggg ggtgaggatg ttagcttatt 3060 tcagtggttc agagggtgcc aggcgaggac ttaggggtgg gcacttgcac agctccagat 3120 aagaaccaac tggctccact gctgtccacc ctaccactca gccattcctt ctggcagctg 3180 ctgtggccat ctcctctgtg tgggttcctg tgctggaacg gagaagagaa agaataagac 3240 ccatacctgc tccagtgtag ggctggagca caactcacct ccgtggaggg caggggatgc 3300 atgctccaga ggtgacccgt actattgctg gagggcagtg gcataggacc aggccttcag 3360 gaggtggccc cggagctggg ccttggagaa tggcttgggg gtggagggat tacagcagca 3420 cagcctgggg ctgagaagtc cataatcgct gcccactagg ccagcatgtg ccagaggtgg 3480 acagggggta ctggggacca ggaagctgtg agtgaaggaa caagaatacc aggttaggat 3540 acctggacgt ggctctgtag gaggcattag gacaagggag tgggccactc agacaaacta 3600 gcaaagcctc agtggcaaac tgcttaacct cttggagctc gcatttcttc ttctgtaaag 3660 aggctgctgt gagacgcaaa ggctgtaatg ggagagcaca ttacttttag gaatagtcat 3720 aattattatg ctgctgttgc tgccgctgcc accaagttgt tgttgttgtt tttgagacaa 3780 gttctggctc tttagcctag gctggagtgc agtggcgcaa gcttggctca ctgcaacctc 3840 tgcctaccag cctcaagtca ttctcccacc tcagcctccc aagtagctgg gactacaggt 3900 gtgcaccacc atgcccagct aatttttgta tttttcgtaa agacaaaaat gttgcccatg 3960 ctggtctcaa actcctgagc tcaagtactc cacccgcctc gacctcccaa actactggga 4020 ttacaggtgt gaaccactgt gcctggcctg agttgttatt ttggcattaa ccctgtgtcc 4080 ttgaaacagt cacctccttt tgaagacctg agatttccct aagatgagat gatgggacaa 4140 gatcagtgtt cttaactttt ttctttttct tttttttttt ttgagatgga gtctccctct 4200 gttgcccagg ctggagggca gtggtgggat ctcggctcac tgcaaccttc acctcccagg 4260 ttcaagtgat tctcctgcct cagcctccca agtagctggg attacaggca cctgctatca 4320 tgcccagcta atttttgtat ttttgtagag actgggtttc accatgttgg ccaggctgtc 4380 ctgacctcag gtgatccacc tgccttggcc tcccaaagtg ctgggattac aggcatgagc 4440 caccacacct ggcctgttct taattttatt ggtcctgagt cacttagaat ctggtgacag 4500 cagtgagccc tcatcgtaga catatacatc catgcacaaa aggtatattc tactctaggg 4560 gatcatggat tcccgtagtg cacgcaggac cccagaaggt taagttccct gtgctagagg 4620 ccctctgagg cccgggcagc tgggaactgc tatggttctg ctctgagttg tctgaacttc 4680 cccctgcctg gatgcctccc tcccatgcag ctccttccct tgctctaggc cccactactg 4740 cctctgccag aggttccagg gctgccactt tgggtagaat gattcctctg tgcctcctct 4800 ccgagaaagt gaagggagtg agccagggcc taaactgtgg tacacttgtc caggaaaggc 4860 ctcttctcct tgcagtggag ggatggcacc aatgtcccca gcttctgcag catcagaggg 4920 ctcagtcccc catctcctgt ccagcctgct gggctccctg gggtctccct gcctctttca 4980 gcctgcctag gccccactgt aagctgctta ggcattaatt aaacccaccc ctgagcctgt 5040 gcttaatggc ctctccagcc gggactgccc agtaaaccca ccaatagtgc ctgactgggg 5100 cgtaggagga ggagcaagcc cagcaatttg gcttcgttgt tgcagaccac ttgatggggg 5160 cgtggtgggg gaggatgggg gacagggagt ggagacttgg ggtgtggcgg tgggttctgc 5220 ttcctcaaag gactcctggg gctcctgctc cccttcatgt tgttcgaggg gctgctcctg 5280 gcttctcata ttcaaaggaa gaagtacaaa gtgtttttct tccttcaggc agcctctgtc 5340 ctatcctgag ctacattagc tcccagcttc aaaaatctaa tggtggattc tgccccttat 5400 tatctggtgg tcattatact tcccacagga tagagagaca gctgtgtccc tgcctctgag 5460 ttcaggctcc ccttccctcc tcctcccctg ggagatccct gccttggact ctgcaaaggg 5520 caaagctaat aagatccttt ttccgccctt ccccatgggt cagtatggtt gaagggaggg 5580 aaagggtatg ggtgtattcc tcttgcaggg agaccccata tctactttgg gtagagtcag 5640 gggccctttt cctgagactt gggagataaa tggacagtgg cactgggctg agaacttaga 5700 gatcctgaac ctggcatctc ctgacctgca gccttgctcc ctcttcccct gtcaccagat 5760 tccctccctt ttttggatca aggctgggat atgtggctgc tttcaggtgg agctgtccct 5820 ttaaatctct cctccatccc tgggttactg tcctggtgtg taattctact gtcatggcaa 5880 ccaggggtgc aatgcgacct ggtgccactc ctcagggaat gtctgagtgg aggctgcagt 5940 accctgatgg tggcctcttg ggcacaagat ctcagaggaa tggggtcacc ggagcggagc 6000 tgagtccttc ccttctgccc acacttttcc ctgtgtgcaa atgtgccctc atttttcctt 6060 tctggggaac tgagttccaa gtatgcctga cttgcttcct gggcatcatc cccttcatta 6120 agaggaagaa ctcatgggag atgggggagg ggaagaagct gctgtgaggc gaggggtaag 6180 cagggctggg gcaccatccc tacccacagc gatgccacct cccccacgta ccctgcctcc 6240 cagtggctga ccaagggtgg gtagggggag gcgtctgagg cttgaggtat ttgccccatg 6300 cccaagtgag gctgcctccc ggtggagggc tgagtcgttc ttggggactt ccttagactg 6360 agggactatt gtaaaggaaa cagggcctgg agaggggact gagagcagag gtcagagggc 6420 atagggagga ggtggcatgg ggtgtattag aggaggggct tctagaaaag agggtacagg 6480 gacaagatgg gccaagactt ttctccctcg ggtctcctga agctggctgt gaggggaggg 6540 tctcggagta ccgcgccagt cttgaagggg cagcatgcct accccctcct caaaacttcc 6600 tgttagccgc ctcctcccca tccccaccga cactgacatg ctggggatcg ggcaatctgt 6660 gggactaggg ctccctccca ccactcttct cccacccggg ttaccgcctg ctctgcaccg 6720 cgcagcgcca gtcagggctt tagccaatca gcaaggcggt cacggcggct gagccttccc 6780 attggctggt tcctcagagg gcgctcctcg actcccgcct tccccagccc ggccaggctt 6840 tggctgggga ggcggtagac ggcggcgtgt aggacgctcc gggagtcccc gggtctgggg 6900 gccgccatgg caggcagccc aggcagcggg gtctccttgg agggtatatc cctggagtct 6960 tctgaggaag cggagctcca gagggaaggt agggaagggc gggccagatg ggggcggggc 7020 agctggcccc ttagcctacc ttcctccagc agagtcgggg catctgggga gggtggcctg 7080 ggagaggtca tctgagggca ccttcaggag agctgtcctg ggcagagggc accaactccc 7140 ttggctcagt cctcccctag ataacggagg gaatctggca taccctggca ggaagggaag 7200 ggtccctgcc ctggcctctt tcagagccca ctgagggcct cgcaggtctc tgaccaatgc 7260 caagccccgg cctgggcaag aggggtctga agggcaagct tgcacttttt tttctggaga 7320 aggcagggag caggaggagg aaggagatgg gagtccagga gtccctgaac catctctgcc 7380 agctccagtt ccagtgcccc ctcctcccca ctcctgttcc ttcctggagg aggggggcac 7440 tgacaagagc attgagaggc gccaggggtg catcctccgc cgtcctccgt ggcttgggta 7500 ctgttatgca gatgtaggca tgggcggcat ggtggcagag taatgatacc cttcgcccat 7560 ctctgccccc agctgggcac attctgcttc tcgcttcctt tccccggaac ttgccacagc 7620 ctggaactcc cacctctgtc agtgagtgaa ggcagccctc acctcaggtc tcgggggcat 7680 tgttgcggcc cagctgcagg tgttatcgtg tcctccatta tgtcccattt cgggctgaga 7740 gtttgcttat ccctgtgatg acttggtgtg acctcagcct ttgggtcaca ccaaggcctc 7800 cagtgcctgt ttccctagct cctgccagcg ttccaggcct cagctttgac ttcctatcat 7860 ccatgttaat ttctcagtga ttatagatca ttgtcactca gagctgaagg cctgtaaaca 7920 tcatctagcc caatccatcc attttacagg caggaaagac tgaggccagt gagggacagt 7980 gacttgtaca atgtcacact gaattagtgg tgcttagact ggaggctgca ggccctggct 8040 cctgtgtggc tgcctgcttg actctgggcc tgtactcacg gacttgctcc gagtactgtc 8100 tgtctcatcc ttgagcctgc caggtacagg tggctgagcc ctgggcttca gcccatctag 8160 aggttgagtg aggagcttgt ggttttcttt tctttttctt tttttttttt ttgagacgga 8220 gtcttgcttc tgtcgcccag gatggagggc aatggcagga tctcagctta ctgcaacctc 8280 cgcctcctgg gttcaagcga ttttcctgcc tcagcctccc ggtagctggg attacaggcg 8340 cctgccacta cgcccagcta attttttgta tttttagtag agacagggtt tccctgtgtt 8400 ggccaggctg gtcttgaact cttgacctca ggcaatccac ccgcttcggc ctcccaaagt 8460 gctgggatta caggcgtgag ccaccgcgcc cggccagaga cagtcttgct ctgccaccca 8520 ggctggagtg cagtggcgtg atctcagctc actgcaatct ctgcctcaca ggttcaagca 8580 gttctcctgc ctcagcctcc tgagtagcta gaattacaga cctacaccac tatgcctggc 8640 taattttggt atttttagta gagatggggt tttgccttgt tgcccaggct ggtcttgaac 8700 tcctggcctc aagtgatcca cccaacagct tgtggttttc tggtgcaggg caggatctgc 8760 aacctgacac ctgcttccct ttctcccttc tctccacctc agagtctctt ccagctgcct 8820 ggagcctccc acctgctcag gctggcatta cctagggtgg agagaacagg ccaaggaaac 8880 tgtccccttc ctccaggatc cctgtcccag gcattcaagg gcctggggtg cctgtgctgg 8940 gcagggaggg gaatgttgct ggggagggga atgttgccgg ggaggggctg ccttctgtgg 9000 gacatggggg tggggaagtg gcaagtattg tggtgtgttt tggtcttctg tacccagctg 9060 gtacctgtgt atctcactgc ccgtaccttc ccccagccat gcagcaagtc ctggacaact 9120 tgggatccct ccccagtgcc acgggggctg cagagctgga cctgatcttc cttcgaggca 9180 ttatggaaag tcccatagta agatccctgg ccaaggtaca gtgctccagg gaggtgggca 9240 caagggcact gtgcttgggg aggcacagta ggggatgggg tattagctac agagggcttt 9300 gggaatggga catgtggcac aagaggcccc cctgaagggg attgtagaag aaaatcatgc 9360 ccaggtgatg acctgagtgg ctgcaggtag agggggctgc ccccacacct gtctggtgag 9420 tgcgtgggtg gatgtttgtt gactgccagc ttgtggagtg tgtgattggt gcgggctagc 9480 atgcacatgt tggagaatct ctagaattca tcttgcctgt ccttctgcct ttgggcgttt 9540 tgtgtgtatg tctttctaag gtcacctgcc atgtatcagg ttggggagag attcactaaa 9600 agagaaaatt ataggacaat ttggtaatga cagtgataga ggtggataca ctatgcattt 9660 tgggagcata aaggacaggt gtctaaccca gcctggggtg ggtgaggagg gcttcctgga 9720 ggaaggacca cttgagctga gtggaagaat gaggaagagg gggagggcat tctgggccac 9780 agaagagctt gagcagaagc acagaggtaa gaaagagtca ggagtgtgca ggggattgga 9840 agggctggtg tgaggctgag cctcaagtgt aagacaatgc gagctgtgca aaggttggca 9900 ggtctggagg gcactggggg ctgtatgtgg gtgcttgggc atccttctgt atcagggagg 9960 ctttatggtt ttttttgttt tgttttgttt tgagaaggag ttttgctctt gttgcccagg 10020 ctggagtgca atggcgtgat cttggctcac tgcaacctcc acctcctgag ttcaagtgat 10080 tcttctgcct cagtctcccg agtagctggg attacaggca tgtgccacca cgcccagcta 10140 attttgtagt tttagtagag atggggtttc tccatgttgg tcaggctggt ctccaattcc 10200 tgacctcagg tgatctgcct gcctcagcct tccaaagtgc tgggattaca ggcatgagcc 10260 actgtgcctg gcaggcttta cattatttaa gcagggatat gatggccaaa tgtgggtgtt 10320 ttggcagcca tgtggaggac tgttgggggg ttatatctga agttggagag gccagttagt 10380 ggctcttcag tggattgagt ataagatgct gagagcctga aatggagctg tggtcccaga 10440 ccttagggta aactggtgga acttggtgtt tggcagtaag ggctagggta cagtaaggca 10500 cctgggatcc tgccccaggg agtgtggaag gatgccccct ctcctgaggg ggggaggtgg 10560 ggatggtgcc catggctgcg ttggaggcaa ggctgggagc tgtgggaaat cagacctaat 10620 tgctcagatt tcttggtgaa ggtggagcag tcctctcctg ggaattaggg atcagagatg 10680 gagcaggaag attcagaagg gaaggtttga ctggtcactg aaagggagaa ggaattaaat 10740 ggatggggac ctcgactgtc catgtcaggt gcccgatttc ccagggtggg ggtgactcca 10800 tccagagagg aagagaaagg agcgggggct ttggtccagg gcacgtgctg gcctggggct 10860 gtggaagtcg gatgcaggga attgtgaagg gagctggtga gagaggctca gagaaaagag 10920 gcccaggctg gaaagaggca gaacagggag aaaatggagg tgaaggctcg gtggctctct 10980 gaggtcacag agtatttgag gggtgatggt agggatatag atggaagaga aaaagaccaa 11040 ggtcagagga agggaggtag gaggtgaaag ttttccaagg aggacagttc tagggatgcc 11100 atgataatgt agggtggggc tgcagtggac aggtaggtca tcagtgttga agacacccag 11160 aaatggaagg ctcaggagtg ggatgggctt gtccacacac ctattggcat gtagggctat 11220 ggcaggcgtc agggtgggga gaagactgag gcagtcccca gggttttcag tgcatggtgg 11280 tggaggtgac caggaagtcg gcaatgacag caagggtagg gagaaagaag gtacagccag 11340 gtggtgggtt ttctgtagag cggggactct gtcacaggct ggctatggct ggccctggca 11400 tgggatgtgg aggaccacgt cccactggag tggggattgc tgctgcaggg gacaccatgt 11460 ccttgggttg tagcctattc acattatctg tggaatggtt gaagacttgg gaagattgtt 11520 aacagcgaaa gatgaaaagt tgcagaggtt taatagtggg aagttctggg gaagagaaaa 11580 ctgagcagat tggaggagat tggagaaagg cagggtgctt ttcattcagc aatcgaagat 11640 cctactcatt tactaaagat aaagacaaga agctgggagt aggggcatgc acctgtagtc 11700 ccagctactc tggaggctga ggtgagagca ttgcttgagg ccaagaagct caagtgcagt 11760 tcatgtaaca tagtgagagc tcgtcttcaa taaataagta aataaataat aacaattata 11820 cacatttcaa acatagagac ttccatgaag gcaggaatgt ttgtcttttt gattcactgc 11880 agtatcccca gagcctacaa aatgcctgac acatagtagg tgctcactga atatttgttg 11940 aatgaataaa tacaaaagag cataaaaaaa taacaaactc ctgagtaccc atctccaaaa 12000 tgttaataat gtttgttttg actcttgcca tttcttctta agaaataaaa agtatgaggc 12060 acacggcacg gtgtggctta cagctttagt cccagcactt tgggagacca aggtgggtgg 12120 atcatttgag ctcaggattt caagaccagc ctgatcaaca tggtgaaatc ctgtctctat 12180 aaaaaaatag aattagctaa gcatggtggc attcacctgt agtcccaact actcaagagg 12240 ctgaggcagg agagttgctt gagctgggta ggtcgaggct acaatgagcc atgatcatgc 12300 cactacagtc cagcctgggt gaccgagtga gacactgtct caaaaaaaaa aataaataaa 12360 taaataaaaa gtttggcaca gttaatgcct ctccccttcc tcattttcac tgcattccct 12420 tcccacaggt aaccactgcc tagagattga tgtcatttca ttgaatgttg gcttatcttg 12480 cctacatatg tgtttagctg taaacaatat acctttgtgg gatttttttt tagacagggt 12540 cttattccat ctcccaggtt ggagtacagt ggaacaatta tagctcactg cagcctggaa 12600 ctcctgggct caaatgatcc tcctgccctc ctgccttagc ctcctgagta actggggcta 12660 caaatatgta ccaccatgcc tggctaatct ttaaattttt ttgtagaggc agggtcttgc 12720 tacattgtcc aggctagtct caaactcctg gcctcaagta atcctctcag cctcccaaag 12780 cactgggatt ataggcatga gccaccatga ctggctctac ctttgttatg tgtgcttttt 12840 tttttttttt ttttttcctc tggagtctca ctctgttgcc caggctagag tgcagtggcg 12900 catttcggct cactgcaacc tccacctccc aagttgaagc gattcttctg cctcagctcc 12960 ccgagtagct gggactacag gcatgcacca ccatgcccgg ctaatttttg tgtgtgtgtg 13020 tgtgtgtata tatatatata tatatatata tattttgaga tggaatctca ctctgtcact 13080 gggctggagt gcagtggcac aatctcagct cactgcaacc tccgcctcct gggttcaagc 13140 gattctcctg ccttagcctc ccaagtagct aggactacag gcatgcgcta ccatgcccag 13200 ctaatttttg tatttttagt agagatgggg tttcaccatg ttggccagga tggtctcgat 13260 ctcttgacct cgcaatccgc ctgccttggc ctcccaaagt gatgggatta taggtgtgag 13320 ccaccgcacc tggccatttt tgtatttttt tttttttttt tagtagagac agggtttcac 13380 catattggcc aggctggtct tgaactcctg acctcatgat tcgcctgcct cagcctccca 13440 aagtgctggg attacaggcg tgagccacca tgcctggcca ttacgtgtgc ttttaaattt 13500 tatataaatg aaatgatagt gtaaggtgct tctgtttttg agatttctat ttcatacaag 13560 tagtacagct atcgacctaa gtatagatat agatagagat agggagattc atttgaactg 13620 cggtgtggtg ttctgataca gcagtcccca acctttttgg caccagggac cagttttgtg 13680 gaagacaatt tttccacgga tcagggtgag gggggtggtt ttggtatgca actgttccac 13740 tcagatcatc aggcatttgt tagacttctc ataaggagca cacaacctag atccctggca 13800 cgtgcagttc acggtagggt tggtgcccct gtgagaatct aatgccacca ctgatctgac 13860 aggaggtgga gctcaggcag taatgcttgc tcacctcctg ctgttcacct acttcctaac 13920 aggccacgga tcactactgg tccatggctt gggggttggg gacccctgtt ctaatatatg 13980 aatagagctg tttgtttatt ggttcccttc ctgagagatg tgtatgtcac tgctgatttt 14040 gctgtcttaa aaacagtgct gcagtgaaca tcctcatatc catctgcttc tgcatgcata 14100 tgagggtttc tctagaatgg acacttggca atgaaattga tgaatatgtg cagttttgaa 14160 cctcaacatc tgtaactttc caaaattgat ttcagagttg ggtttatgag cccacatcac 14220 ctctgggtac tttagctatt gtctccacag tgagacagtg agactctgct ttgtgatggc 14280 agagtctcag aatgagacag ccctcttgcc aatacccttc aaagcagggt cccccttgat 14340 agcattcctc tctggtccag tccactcgac agtccactcg acagctctct aatgatgggt 14400 ttgctggctg gctggctatg tcattcactc atttgtccat ccatccatcc atccatccat 14460 ccatccatcc atccatcaaa tacctatttt gcactcgcca cataccaggc acagtgctgg 14520 ggttatggca gtgaactgcc agatagtttg ttttcctgat gaagcccaag tctgtcttcc 14580 tttcactgga caccacaatg ccccagtcct gggacctcac agtaaagcct aattgaaaat 14640 gaaccaatgt gctgtgtcac attgtaagta aactcccttc tctggaaacg tttttggcga 14700 gaagctggtc aggatttttt ccaagaaatt cctggtatgg ttccggattt gtagagcaca 14760 atagaagaga gtatggacgt ttcttcccct ctgagacccc cagtatgagg ctaatgggct 14820 aaggcaatcc agtaacttct ttctccctgt ggttgtgaaa caaacaggaa gtgaatctcc 14880 ataaaggagc ctacctgagc ccactggaaa aaaaaaagcc attcatttct tctgtccttg 14940 tatgggtgag cctttgcaat gtaaatttgc cactcctctc atcaaagaca gtgtctctca 15000 gagctccctg cattggacaa tgggacctac ttcctccttc tcaccaattt taatcattat 15060 gtgtacacag acaaatttct aacctaatag acaaagcact gtgatcaaag atccagctgc 15120 aaaaacttcg tgaagtggcc cagaaaccca caggaatgaa atcagattgg agccacatgt 15180 ggtagctgtt ggcctccctc ccttacgctg tggagctggg cttgactctg tctggggatg 15240 tggcagacag tgtaggaggg gccaggctgg gtagatctgg gaccagggag ggacagggcc 15300 tgaggataca aacccagagg gttaaaggcc ctaagggaag tcaagtctgg cagagagatc 15360 agaagtccag gcggttggct gcacacagca agtggcggcc ctggcaggtg gctggcatct 15420 gggacggctg acatggatcc agtagcaggt gttccagcta cagacaaggc caaggtgagg 15480 aatccaaggc aggggacatt cagaaattca gaaaaattgt tactaatggg gacagctgag 15540 aggcatgtga aggctgttgt gaactaattc acatgagatg aagaagaaag aacacagggc 15600 ttggagtcag aagattccac tgaccagctt tgtgagtttg gtatatctgt caacctctct 15660 gaacttcagt tatttcttat ggaggaattt ttaaaaaatt aatatttctt tatgtattta 15720 ttttaaaaga aaggtgaggt ctcaccgtgt tgcccaggct ggtcttgaac tcctgagctc 15780 aagtgatcct cccaccctgg cttcctaaag tgctaggatt acaggtgtga gccaccatgc 15840 ccagcccagt tatttctttt gagaagtggg gagaatgata cctatccctc agagtcgtgg 15900 tggggctagg tggaggttat ggaagtgtaa gttttttttt tttttgagac ggagtcttgc 15960 tttgttgccc aggctggagt gtaatggtgc aatctcggct cactgcaacc tctgcctccc 16020 aggttcaagc gattctcctg cctcagcctc ccgagtagct gggattacag gcccaccatc 16080 cccggctaat ttgtgtattt ttagtagaga tggggtttca ccatgttggc caggctggcc 16140 ttgaactcct gacctcaggt gatccaccca cctcggcctc ctcccaaagt gcaggaatta 16200 caggcatgag ccaccccggc cagaagtgat ttttgaatca ggaagagttg ttataatctt 16260 agcttcagac tagagttcac agggaggcct cttgttctgt ggggaggcta gggtggaaga 16320 cctgggaaag caagacaggg ccctagaaga ataaaggtct agggaggtca ctaagatagg 16380 ggctctgaca aggggacatg acaagcagag ggtgtagtgg tgaggccagt gtatcagttc 16440 acttttgctg tgtaacaaac catcgtgctg tgtcgtggct taaaacaact gtgatttaat 16500 tcccctgatt ttgtgggttg gttgggtggt tcttctggcc tgggcttgct tagctggggc 16560 tgggtggtcc gggatggcct cactcacatg tctggtgtct tagctgggaa aactgcaatg 16620 tctgggatgg ccagaccccc tttccatgtg gtctctcatc ctctaggagg gtctctcatc 16680 ctcagccacc ttcacatggt agtctcacaa gcaaggaagg acaagcctcc agtcacaagc 16740 actttctaag cctctgctca catttgataa tggcccattg gctaaagcaa gtcacatctg 16800 tcaaacccag attaaagagt tcctttactt tctgacacac tactctctgg gacatctttt 16860 aatttaacta tttaattatg aaccagggct tcaaatagga attccctgtc ttttgccatg 16920 atcggggggt tggagtggag ggcagaggga aaacttttca gtgaaatgac tgtgtagtgt 16980 ttgcatttgc agcctgctac agctctggat tgggagcaaa cagaacagag tgcaggctgg 17040 tgccactata aactagctgt gtctcccact taagcttctg gtctaccatg agcttgagcc 17100 cctgctttgc tgaggataat tttcagagca gaatgaaatg aatggctacc acagaaattc 17160 aaatcactct tctagttctg ggccatgtgt cacttaacat gaaaaagaag aataagtctt 17220 agcaaaataa tagtgagtaa aaaagtagta agggaaataa ttgttcactt aatgttaagt 17280 gaaaaaaaat cagaccccag ggatggataa aatcatgatt cccccctttt tttatgtcat 17340 agaaaatacg gccgggtgcg gtggctcacg cctgtaatcc cagcactttg ggaggccgag 17400 gtgggtggat caccagttca ggagatcgag accatcctgg ctaacatggt gaaaccccgt 17460 ctctactaaa attacaaaaa attagccagg cgtggtggtg catgcctgta gtcccagcta 17520 ctcgggaggc tgaggcagga gaatggtgtg aacccgggag acggaggttg cagtgagccg 17580 agattgcgcc actgcactcc agcctgggca acagagtgag actctgtctc aaaaaaaaaa 17640 aaaaaaaaaa aaaacgcatt tggacagaca caattccctt tttattaact atattcattt 17700 cagaatttca gaagtatgta gaatacatac agacacatta caactcttta actgggaagc 17760 cagggaatga caaacacact aaatcaagat ggtgctttca gctgggcatg gtggctcatg 17820 cctgtaatcc cagcactttg ggaggctgag ccgggcggat cacctgaggt ctggagttca 17880 aggccagcct caccaacatg gagaaacccc gtctctacta aaaatacaaa attagccggg 17940 tgtgttggtg catgctgtaa tcccagctac tcgagaggct aaggcaggag aatcacttga 18000 acccgggagg tgtaggttgc agtgagccga gattgtgcca ttgcactcca ggctgggcga 18060 tagagcgaga ctctgtctca aaaaaaaaaa aaaaaaaaaa gatggtgatt tctgcaagtg 18120 ggaagggtga gtggggcatg tggttagatg taggatattg acaagagctt tgttttttag 18180 tttgggtggt gggttctctt agatccttgt tatatcattt aaaaaatcta atttaggcca 18240 ggtgtggtgg ctcacgcctg taattccagc actttgggag gccgaggcag gcagatcacc 18300 tgagctcagg agtttgtgac tagcctgggc aacatggtga aacctcatct caactgaaat 18360 ataaaaaatt agccaggggt ggtggcgcgt gcctgtagtc ccagctgttc gggaggttga 18420 agcacaagaa tttttgagcc caggaagtgg aggttgcagt gagccaagat tgtgccactg 18480 cactccaatt tgggctacag agtgagactc cgtctaaaaa aaaaaaaaat tatttgttca 18540 gcctactgaa ttgttggttt tgtatttgaa tgattaaatg tttaaagttt cctctgggct 18600 atgaggttga gcttggagag ctgtgctttc atatctgtgg gtataaaacg tccaggggtg 18660 gccacaggag gaggaatgga ttagcccatg gtgggaatgt cattaagtca aagataggag 18720 gtaagcagct taggaggcac cctggcttcc cccttgatca cacacgtctc ctcggaaaac 18780 ttgtcctcca tccgtggacc agatagtgct aaagtcaaaa tgagtgactt ctctaatgct 18840 cacatcagga cacctgtgcc agagctggct cctgttaacc caaaagagcc atgcaagggt 18900 tgccatggtg agattgttgc tgcctgagtg ccattgttta ttcttgacac tcagttattt 18960 gggctttgat tccaacattg tcctaggcac agggtaatag agccagagct gggacgcctc 19020 cctcactctg gggctccata ggctgggcca agctaaatga ctgaaagtca cataggggtc 19080 tgtggaagac cagccctctg gggcttgttc ctttccccaa atgctactcc tctgccccat 19140 cagttgtctg tttccccagt aggttgtgga tgtcagtcta aaatgctgcc cacccggcag 19200 aggatggtat gggaggctgt ggggccatgt ggggtgctgt tccctggcct gtttgggaaa 19260 agagaaggca gaaagcatct aacctttatt gagcacctgc tgtctgccag gcactatgtc 19320 catctcacca aatcctcaca gcagtcttat gaagtattac tcaaatctca ccatctcatg 19380 gctaaacaaa ttttctatta tgaaaaaatt taagaatata tcctgaacct ccatcaccta 19440 gcaccccact tcaacattca acaagtcatg cataatcttg tttcatccac acctttacca 19500 gcttccctct ctcctccatt gttttggagt acatctcagc cataatagat tgtatctgca 19560 agtgtttcat tgtgtatata aactcctggg ctcaactgaa aaggattctt taaagctaca 19620 ctactattct cacacctaaa aaaaaatcaa acaatagttt tcttttgaat ttttttaatt 19680 aaaaaaattt taaaaaatag agatggggtc ttgctgtctt gaactcctgg gctcaagtga 19740 tcctcctgcc ttggcctccc aaagtgctgg gattacaggt gtgagccatt gtgccctgcc 19800 atgaattttt aataaatggt attggaacat ctggatagtc attttgaaag atatcaaagt 19860 gtatttattc ctcacatcat atatcagaat aaactccaag cggatcagag atctctatgt 19920 gaaaatataa atcatacacg tcctagaaga aaacaaggct ctgtatatgg gaaaagccct 19980 cccttaaaaa aaaaacaaaa aaacaaaaaa ctatgcctca aaattcagat gcatgaaaga 20040 ttattaaatt cacccacatt aaaaaatttg ttttttgagc cagggtcttg ctctgtcact 20100 caggctggag tgcagtagtg caaacagtgc tcactgcagc ctcaatctcc tgggttcaag 20160 tgatccttcc acctcagcct cccaagtaag agtagcaggg acatcaggtg catgccacca 20220 tgcattaaaa tgaggattaa aaatgctcag gtgaattttt aaaatatttt gtagcaacag 20280 gggcttccca tcttacccag gctggtcttg aactcctggg ctcaagcagt ccttcctcct 20340 tgacctccca aagtgttggg attgcaggtg tgaaccactg cacctgacct aaattttttt 20400 tttttttttg catgcaaaaa caccacaagc agagtcaaaa gacaagtgac aaactggggg 20460 aaatcattta caagaaatat cacaaagggc taattttccc aatagagaga aaacattttt 20520 taaaataggg aaaagaataa aaaccaagac ctgacagaaa aattgggaaa gacatgagta 20580 gacagttcac agaaaaagat atacagatgg tccttaatca tatggagaga tgtttgactt 20640 tactcataat aagcaaaatg caaagtaagc cccaatgaga gatcatttct cctccatgtg 20700 attggcagac atgcaaatgt ttgatcaaca ctctggtggt gagcttcaag aaaatccctc 20760 atacagtgct agcaggagag caaactggcc aaagtcccat ggaggggaat ttgtcaatat 20820 ctaagaaaat tacgtatgta tttgctgctt gacccagcaa gacttctagg aatttacatg 20880 gaagatcttc tccacagata tagaatacta tgccggagtc atttactgca acattatttg 20940 caatagcaaa gtattagaaa caacctaaat acccatggag actggttgaa taaattacag 21000 gaatccagtc atgcaatgga gtcaaatgca gtaattaaaa taataagatg ggcatggtgg 21060 ttcatgcctg taatcccaac ctgggaggca gaggttgcag tgagctgaga tcgcgccact 21120 gcactccatc ctgggtgaca gagcaagact ctgtctcaaa caaactaaca ataataatta 21180 ataaaggaag atctccatgc agtttcatgg agtgatttct atgacatatt tttaaataaa 21240 aagccagaaa accaaaccag ttaaataaaa gccctagggt atcaaagggt atatacagtg 21300 ttactgtgtt ttatgtaaga aagggcaata tatttattta attttttttt ttggaaacag 21360 agtcttgctc tgttgcccag gctggagggc agtggcgcga tctcagctcc ttgcaacctc 21420 tgcctcccgg gtccaagtga ttctcatgac tcagcctcct gagtagctgg gattacaggc 21480 atgtgccaca tgcccggcta attttgtatt tttggtagag atggtgtttc accatgttgg 21540 tcaggctcgt ctcaaactcc tgacctcagg tgatcctcct gccttggctt cccaaagtgc 21600 gggattacag gcatgagcca ctgtgcctgg cctttagtgg tgatttctga gattttgatg 21660 cacccatcac ccaagcagta tacactgtac ccagtgtgta gtcttttatc ccttacgccc 21720 ctactaccct tcccggctga tttttactaa tagatttgtg ataggtagta gcagttgtat 21780 agcaattttg aaactgcttt gaatattgaa ggacagaaca aataagtaaa tatactgatg 21840 ttattgggaa ataagttctc actgaaggag aagggagaca caaatatgga atgggggaag 21900 atgagaaaaa tcctttaatt ttatttggag ccattagtat ctactaatga ctgccaaaga 21960 aatgatgccc agaaacaatg ggtattgagt gtccacatct tggtttctaa atactatctc 22020 ctgctaaaag gaatcagggt tccttggaga aattgctgat tccagacctg gggtagggaa 22080 tgcatcagat gagcctgtgc cagaagggaa gaagatgctc aaggactgat gggaacatgt 22140 caacaggaca gaggtgtgaa tggggctccc actggacaaa tttgggataa actgggtcaa 22200 cgaagatagt gatcgtaatg gattagaaca catggaataa aaaaaattca tagtgatatc 22260 atgagagaga gaaggagaga aagaggagta aggtggtagg gagaaaagaa gagctcattc 22320 tttttttttt ttttttttga gctggagttt tgctcttgtt gcccaggctg gagtgcaatg 22380 gcatgatctc ggctcattgc aacctccacc tcccaggttc aagcggttct cctgcctcag 22440 cctcctgagt agctgggatt acaggcgccc accactatgc taggctaatt ttttatatct 22500 gtagtagaaa cggggtttca ccatgttggc caggctgttt tgcaaactcc tgacctcagg 22560 taatccacct gccttggcct cccaaagtgc agggattaca ggcgtgagcc actgtgcctg 22620 gctgaaaagc tcattcttat agcagaatgc caataaatgt agaaggaatg atgggaatta 22680 ggtaatcact attttgcaac ctccagtata ataactcatt taggcaaggg tcataaattg 22740 acactaaatc cattggtgaa ggattttggg gtaacagaat agtcacatag tctcaaagta 22800 tgggaaagac acaaagacac aagatcacct acgtagaact cttgtcaaaa ggattaacgt 22860 aagtctaatc atgaggaaac aatcaactaa atctagtggt agagcatcct aaaaactact 22920 ggcctggagt ctaaaaatgt ctaggctggg tgcagtggct catgcctgta atctcaggct 22980 ttgggagcct gaagcaggag gatcacttga agccaagagt ttgagactag cctaggcaac 23040 gtagtgagac cctgtcccta caaaacaaaa acagaaacaa aaaaaacaaa acaacaaatg 23100 ttctgaaagt ctaaaagtta ttgggatctg ttctagatta gaggagaata aaaagatatg 23160 tcaaccaact acaacatatg atcatcaatt agatcctgaa tcagaaaaaa taaaattata 23220 aaggatactt ttgggacaag agtgaacatt tgaatgtgga ctgtcttaca taatgtatta 23280 atgaatcgat gttaaatttc ttgcaggtga taatggtatt gtggtttatg cagcagaatg 23340 tctttgttcc tatgaaatac atgctgaaat actttggggt aaagtggctt gtgttgtctg 23400 caatgatctt tcaaagattc agaggggaaa aatggtcttc atatatatta ttgaaatgga 23460 attcttttct tcctgaccca tctaacctaa cactccccac tgggctggtc actggcgaag 23520 gattcctttt ctgttttcag cgctccttgg tatggagccc ggagccagca gagcaacaaa 23580 taggtgcagg gcctgctcct ggcaccgtgt tctgatttcc tgtcccctca ttcgctggga 23640 acactgagtt gagggcctgg agacgcaagt tcaagtccca ttgaccctgg gaaagttatt 23700 ctacctccct aatttcctca tcctgggctt cagggtcttc aaggccccac ccaaccatga 23760 tgtctggtgg aagaagccgt ctttgaacgc caatgcatat agggtcctgg gagctgggga 23820 caatttggta acttctaatg accctctgtg gcaaggggat ggaaaggaag gtttgtcctt 23880 tggtttcttc cttccctaaa ctaggtccta gtaattttgg ggaagaaagg agacagcaag 23940 gaatgcatgt gcacgtgtgg gtatgattgt cagtgagtga ggagctgaag ctgggggctg 24000 gggggtggga tggggaaaat gcctgggggc tgagggagga ccctgcttgg tgtcccaggg 24060 ctcctgggga agctcctagc cccacctgtt ctgcttccac ccccaggccc atgagaggct 24120 ggaggagacg aagctggagg ccgtgagaga caacaacctg gagctggtgc aggagatcct 24180 gcgggacctg gcgcagctgg ctgagcagag cagcacagcc gccgagctgg cccacatcct 24240 ccaggagccc cacttccagg ttcctggctg ctagggctgg ggtgagggag caggaggtgg 24300 gtggactggg gcatgctgct gttggatggg ccagcaggaa gttggatctg ggatgggaag 24360 agacccggga cactgcccca ttgtcccttc tcttcccacc accccccagt ccctcctgga 24420 gacgcacgac tctgtggcct caaagaccta tgagacacca ccccccagcc ctggcctgga 24480 ccctacattc agcaaccagc ctgtacctcc cgatgctgtg cgcatggtgg gcatccgcaa 24540 gacagccgga gaacatctgg tgaggactgg gcagggccag aggtggtgct ggtgagggtg 24600 gggggattga gaataaccaa tgaacggaca aaaaaggcca agtgtggtct gaagatgaag 24660 atggggccag ctttgtgcag ggaaggatcg atgcagcaaa gggtcggggg aagacccagg 24720 agaccataga cactgcacac acacctgtgt ccgcacctct ccagtctgcc cacctctccc 24780 ctcattagta cctgctgtaa gtgaagaatt taggcagaag gatggaggag gactttgtga 24840 gggtgggggt gggtgggtag ggacaggaat ggaggagcta ttaggagact atttgaagat 24900 tctgacttgg agcaattggg gcctcatctt acactccctc tgacctccag ggtgtaacgt 24960 tccgcgtgga gggcggcgag ctggtgatcg cgcgcattct gcatgggggc atggtggctc 25020 agcaaggcct gctgcatgtg ggtgacatca tcaaggaggt gaacgggcag ccagtgggca 25080 gtgacccccg cgcactgcag gagctcctgc gcaatgccag tggcagtgtc atcctcaaga 25140 tcctgcccag ctaccaggag ccccatctgc cccgccaggt gggcccctca cccagcacaa 25200 ggcccaaggg atggctgagc ctcctggctg tcaaggttgc aggtctcacg gaggccctcc 25260 tctcctactg gtttcctcca gggaagatgg ggggtgggac ctgcagccca ggggcagctg 25320 accgcagccg ccgacaggcc tctgccgctg ctgcacctgc acatgctccc acattggctg 25380 gtattatgca cgcagccaca gcctggggta caagtgtatg agagagcgcc tgcattatgg 25440 tcagcgttct tcacgtttgt gcgcgtgcgc agggtggggt tgtcatatgt cctggatagc 25500 tgtctcctgg ttggtcaatt ctgggggtgt ggctgctctg tctccctatc ctattgggtc 25560 tctcgccctc caccagagcc cttctccaag gggagagggt gagtggaggg agggatccag 25620 gattaggtgc cagggtcctg tgccctactt ggtggtcccg catccttggt cctgagatct 25680 gaatcctgta gtactagatg caaagggagg ttggcaccag ggaggagcat gggcaggcct 25740 ggaggcatca ctcctgcagg gggacagttc cattgcccct tagtctctgt gaaaagaggg 25800 ggcatggggt agacctccac ctgcagcctt ctgctgaccc ttcccccgac cctttgctga 25860 ttcctgggcc caggtatttg tgaaatgtca ctttgactat gacccggccc gagacagcct 25920 catcccctgc aaggaagcag gcctgcgctt caacgccggg gacttgctcc agatcgtaaa 25980 ccaggatgat gccaactggt ggcaggtgag ctgcgggcac ccccgcatct ctccaggtat 26040 ggtgcatggg agggcgaggg cacagggagg ggtcctcttg ctcaggcaga tcttggtctc 26100 atgtccgttt taggcatgcc atgtcgaagg gggcagtgct gggctcattc ccagccagct 26160 gctggaggag aagcggaaag catttgtcaa gagggacctg gagctgacac caaactcagg 26220 taggaggtcc cccctacccc acacctccaa tctgggatcc aacagggccc tgtctgcctc 26280 actcacccat ctccctatgg ccagggaccc tatgcggcag cctttcagga aagaaaaaga 26340 agcgaatgat gtatttgacc accaagaatg caggtgggta tcagagcgcc ccctccctta 26400 ccctcctcta acatggcatg cctttccctg tccccaccca tcacctgtga cgacgtgtgc 26460 ctggctcaga tgctgccagc cgtgtcccta ggccctgccc gactcccccc tgctcccctg 26520 ttccaagccc tcttgccctc agcctccctg agagctccgc ccctccctca ccgtgcagcc 26580 tggtgagcag cgccatctcc ctgtgtccag agtttgaccg tcatgagctg ctcatttatg 26640 aggaggtggc ccgcatgccc ccgttccgcc ggaaaaccct ggtactgatt ggggctcagg 26700 gcgtgggacg gcgcagcctg aagaacaagc tcatcatgtg ggatccagat cgctatggca 26760 ccacggtgcc ctgtgagtgg gagctgggcc ctgctgagtt ggggacaagg agcagggctc 26820 ccctggggcc agggctgggc ccaccgaatg ggcatcttca tgggcaccag gccaggtggt 26880 acctttgggt acacgggttg gggtgcacgc cctctgtggg tggttagggg aacttcaggc 26940 tcaggaaggg tgagggaggt gtccagccaa caggtggcct agcgggctgg gctggcgtcc 27000 actttccttc tcttgtttca cttgctaaaa gccatgaaga aagtgagtgc ccagctgtgc 27060 acccgggtga aacaggctac gagctgcatg tgtgtagcct tgtggaagac agggcttctt 27120 agtggcagaa acagcggggg tggtggaagc tcctggctgc cagaggcagg actaagagca 27180 ctgccctggt ctgcttggct cttgctgtgt ggctttgggc tgtacattgc cctttctgga 27240 cctcagcctc tcttctgtgg gctctgggct ggaccatgat taccagctca tgtctctgtg 27300 tgtgccaatg gggagagtgg ggggcaggtg tgtgcaaagg cagcagtgct ggcaagatgg 27360 ggagcagact gcacctccaa ggctgggaaa cgggggcctg ggtaggggtg catatatgtg 27420 tctgaggcac tgtaacctgc ccctgcccat ttggtccaac ctacccccat ccccctagac 27480 acctcccggc ggccgaaaga ctcagagcgg gaaggtcagg gttacagctt tgtgtcccgt 27540 ggggagatgg aggctgacgt ccgtgctggg cgctacctgg agcatggcga atacgagggc 27600 aacctgtatg gcacacgtat tgactccatc cggggcgtgg tcgctgctgg gaaggtgtgc 27660 gtgctggatg tcaaccccca ggtaccgcca ctctgctcct tcccagcctg cccaatgtcc 27720 tctctgctgc ctcattgctc ccccatatag ttccagacac ctgtcacctg aacacgccca 27780 tgtaccccct ttgccctcaa ctactgctag aaacccagcc cacctggaac ctttctcagc 27840 cttccagggt ccccactcct cctctagtct cctccagtca ccccagccac ctgccagctg 27900 tttgtcattt gtcccaggac tcatgtccca gacccccggg cacccttttc tcatccactc 27960 ccaagtgctc accccaaccc cagttaccct cacagccttt ctctggttcc taggacagac 28020 atggggatat actccctacc ttgccttctc cttctgcagg cggtgaaggt gctacgaacg 28080 gccgagtttg tcccttacgt ggtgttcatc gaggccccag acttcgagac cctgcgggcc 28140 atgaacaggg ctgcgctgga gagtggaata tccaccaagc agctcacggt gagggctctg 28200 aggtgtgggg gaggggtctg atagctgcct agggtggtgg gggacaggcc tgagctgaca 28260 gagagaagga ggtggtgttg gggagggaca ggggcctgct ccagttatca gtgaggcagg 28320 acgtggtccc ccctgcatag atgtggaagg ggaggtctga gagaggaagt ccaaagtctc 28380 agagtgagcc aggcacacac ctagcctggg atacatgtgc ttctgactca aggcccccgg 28440 ctcagcccac tctgcagaga cagagtgctg gccagagaga gttcccgagg gcaactggga 28500 gaggtgggct ggaggtgggc caggtgagct tgggttgctt acagctgggg gaaagctgaa 28560 gggtaggaga aagccagagg cgggaaggct ggctggaggg ctgagtcagc gtcagggagc 28620 aggcagcgcc tggaggagag gcttcaaggc ctcattgcag aactccagcc ccatcctcct 28680 tcctatgtct tgctgcgcct tcccaggagg tggagggttt gcggcgctct gagcgcaggg 28740 gtccctctgc tggccatgcc tggtgctgca gcattccggc cccaggtcag agcgtgcctg 28800 gggcccagga gaggctgtct aggctggcac ccttctgcca atgaccccct taaggagctc 28860 tggacctttg cctgatcccc attctgggca cctcctcctt ggcaacaggt gtggccaact 28920 ggagctgttg ggatgcagct agcttcagct tatttaagtc agttgtcaag tacccttcct 28980 cacccctgcc catccagctt ccactgggtc gagggtgggg ttgccctggg tacttgtgga 29040 ttgccctggt accagcctgt ctagctgcat aggtaaggca gcctagtcag acttgaggga 29100 gcactggctt gagagtcagg cagaccctgt ttagccactg actggctatg tgaccttttt 29160 ataccttcat tttctctgct ataaatggga aagaattaat cctatcttgc agagctgtag 29220 tgaagattat gtggcatgca aagcatttag gggtgtgatt ggcagttatt aaagcttatt 29280 tcctcccttt cactcaccat ctgtggttga tggtgagcca ctttgaggca cgcagagagg 29340 ggcccctgcc tccctagcca gggagtccac accagggttg ggctttgggg agggggagtg 29400 ggggcagctg tagctccttc tgacagtggg ggtgagttgt gggcactgac tgtagggagg 29460 tccccttgtg cctgggcagg aggcggacct gagacggaca gtggaggaga gcagccgcat 29520 ccagcggggc tacgggcact actttgacct ctgcctggtc aatagcaacc tggagaggac 29580 cttccgcgag ctccagacag ccatggagaa gctacggaca gagccccagt gggtgcctgt 29640 cagctgggtg tactgagcct gttcacctgg tccttggctc actctgtgtt gaaacccaga 29700 acctgaatcc atccccctcc tgacctgtga ccccctgcca caatccttag cccccatatc 29760 tggctgtcct tgggtaacag ctcccagcag gccctaagtc tggcttcagc acagaggcgt 29820 gcactgccag ggaggtgggc attcatgggg taccttgtgc ccaggtgctg cccactcctg 29880 atgcccattg gtcaccagat atctctgagg gccaagctat gcccaggaat gtgtcagagt 29940 cacctccata atggtcagta cagagaagag aaaagctgct ttgggaccac atggtcagta 30000 ggcacactgc cccctgccac ccctccccag tcaccagttc tcctctggac tggccacacc 30060 caccccattc ctggactcct cccacctctc acccctgtgt cggaggaaca ggccttgggc 30120 tgtttccgtg tgaccagggg aatgtgtggc ccgctggcag ccaggcaggc ccgggtggtg 30180 gtgccagcct ggtgccatct tgaaggctgg aggagtcaga gtgagagcca gtggccacag 30240 ctgcagagca ctgcagctcc cagctccttt ggaaagggac agggtcgcag ggcagatgct 30300 gctcggtcct tccctcatcc acagcttctc actgccgaag tttctccaga tttctccaat 30360 gtgtcctgac aggtcagccc tgctccccac agggccaggc tggcaggggc cagtgggctc 30420 agcccaggta ggggcaggat ggagggctga gccctgtgac aacctgctgc taccaactga 30480 agagccccaa gctctccatg gcccacagca ggcacaggtc tgagctctat gtccttgacc 30540 ttggtccatt tggttttctg tctagccagg tccaggtagc ccacttgcat cagggctgct 30600 gggttggagg ggctaaggag gagtgcagag gggaccttgg gagcctgggc ttgaaggaca 30660 gttgccctcc aggaggttcc tcacacacaa ctccagaggc gccatttaca ctgtagtctg 30720 tacaacctgt ggttccacgt gcatgttcgg cacctgtctg tgcctctggc accaggttgt 30780 gtgtgtgtgc gtgtgcacgt gcgtgtgtgt gtgtgtgtgt caggtttagt ttggggagga 30840 agcaaagggt tttgttttgg aggtcactct ttggggcccc tttctggggg ttccccatca 30900 gccctcattt cttataatac cctgatccca gactccaaag ccctggtcct ttcctgatgt 30960 ctcctccctt gtcttattgt ccccctaccc taaatgcccc cctgccataa cttggggagg 31020 gcagttttgt aaaataggag actcccttta agaaagaatg ctgtcctaga tgtacttggg 31080 catctcatcc ttcattattc tctgcattcc ttccgggggg agcctgtcct cagaggggac 31140 aacctgtgac accctgagtc caaacccttg tgcctcccag ttcttccaag tgtctaacta 31200 gtcttcgctg cagcgtcagc caaagctggc ccctgaacca ctgtgtgccc atttcctagg 31260 gaaggggaag gagaataaac agaatattta ttacaaatgt tagaatatat ttcttatact 31320 aggaatctca tttgcatttg catagactat acacatgggg tggaaaggcc aggcctgccc 31380 ccatctcgtt ggtgtggctc tgcgtatact acacactcat tctcctgctc ctcttttccc 31440 ttagtcagtg tcctttcatc ctgattcagc tctgccttgc atcaccctca gcctaaggga 31500 gtgggaagga aatggggtgt tttcttgctg acctgaggct atagggtcac ttgccatttc 31560 ctaccttctc tgggggattt gagggtagag gcaggggaag atctgttgtt gcagttgctt 31620 ctgccccctt gatccaaatg accatcatct ctgatggaga tgggttgggt acctggcctt 31680 catggcacct tcactgctag ggatgctcaa ggggcaggcc tggggccctt ccctcctgtc 31740 tcttctcggt ctttcctctc tgagcagcct cctacctccc ctgcctgagc cctcactcca 31800 cagccctccc aggtacctag cagaggctgt cagtccttgg ctcacctgga acagggctgg 31860 ggctgggttg gaacaggtgt gtgcccccac cacagctcta tgactctgtt ctccctccct 31920 gccattgtgg actcttgtat ttgagggacc tcaagagagt gaggacccta ccatccactg 31980 tccatattca gtcccagccc cagtgcgctt cctctgttcc ctccctcagc catccaattc 32040 ttgagttttc tcactgattg gttttctttc tttttccttg gattaaatgt gaaagcaaag 32100 gcttctggct ctgcttttct ttggttgggg ttggatggat ggctttggga gagagtgaga 32160 gctggggagc acagcacaga tgactactag aatggaagtc agagcaacat gtcttgtttt 32220 ctgcgatgtc ttcctttcct ctgtcttgcc cctgcacact cccccatccc cagaagatgc 32280 tccttctaag tgcttgtacc caaggacatg aacagacatt tctcaaaaga agacatacaa 32340 gtgacaatgt atgaaaaagt gctttacgtc actaatcatc agagaaatgc aaaccaaaac 32400 taccatgagt taccatctca caccagtcag aatggctatt aaaaggtcaa aaaataagag 32460 atgctggtga agttgcagac aagagggagt gcttatacac tattggtggg aacgtacatt 32520 agttcagccc ccgtggaaag ctgtttgggg agttctcata gaactaccat tcattccagc 32580 aatcccatca ctgggcatct acccaaagga gaagaaatcg ttccaccaaa aagatacctg 32640 cacttgtgtg actg 32654 4 576 PRT Human 4 Met Pro Val Ala Ala Thr Asn Ser Glu Thr Ala Met Gln Gln Val Leu 1 5 10 15 Asp Asn Leu Gly Ser Leu Pro Ser Ala Thr Gly Ala Ala Glu Leu Asp 20 25 30 Leu Ile Phe Leu Arg Gly Ile Met Glu Ser Pro Ile Val Arg Ser Leu 35 40 45 Ala Lys Val Ile Met Val Leu Trp Phe Met Gln Gln Asn Val Phe Val 50 55 60 Pro Met Lys Tyr Met Leu Lys Tyr Phe Gly Ala His Glu Arg Leu Glu 65 70 75 80 Glu Thr Lys Leu Glu Ala Val Arg Asp Asn Asn Leu Glu Leu Val Gln 85 90 95 Glu Ile Leu Arg Asp Leu Ala Gln Leu Ala Glu Gln Ser Ser Thr Ala 100 105 110 Ala Glu Leu Ala His Ile Leu Gln Glu Pro His Phe Gln Ser Leu Leu 115 120 125 Glu Thr His Asp Ser Val Ala Ser Lys Thr Tyr Glu Thr Pro Pro Pro 130 135 140 Ser Pro Gly Leu Asp Pro Thr Phe Ser Asn Gln Pro Val Pro Pro Asp 145 150 155 160 Ala Val Arg Met Val Gly Ile Arg Lys Thr Ala Gly Glu His Leu Gly 165 170 175 Val Thr Phe Arg Val Glu Gly Gly Glu Leu Val Ile Ala Arg Ile Leu 180 185 190 His Gly Gly Met Val Ala Gln Gln Gly Leu Leu His Val Gly Asp Ile 195 200 205 Ile Lys Glu Val Asn Gly Gln Pro Val Gly Ser Asp Pro Arg Ala Leu 210 215 220 Gln Glu Leu Leu Arg Asn Ala Ser Gly Ser Val Ile Leu Lys Ile Leu 225 230 235 240 Pro Ser Tyr Gln Glu Pro His Leu Pro Arg Gln Val Phe Val Lys Cys 245 250 255 His Phe Asp Tyr Asp Pro Ala Arg Asp Ser Leu Ile Pro Cys Lys Glu 260 265 270 Ala Gly Leu Arg Phe Asn Ala Gly Asp Leu Leu Gln Ile Val Asn Gln 275 280 285 Asp Asp Ala Asn Trp Trp Gln Ala Cys His Val Glu Gly Gly Ser Ala 290 295 300 Gly Leu Ile Pro Ser Gln Leu Leu Glu Glu Lys Arg Lys Ala Phe Val 305 310 315 320 Lys Arg Asp Leu Glu Leu Thr Pro Asn Ser Gly Thr Leu Cys Gly Ser 325 330 335 Leu Ser Gly Lys Lys Lys Lys Arg Met Met Tyr Leu Thr Thr Lys Asn 340 345 350 Ala Glu Phe Asp Arg His Glu Leu Leu Ile Tyr Glu Glu Val Ala Arg 355 360 365 Met Pro Pro Phe Arg Arg Lys Thr Leu Val Leu Ile Gly Ala Gln Gly 370 375 380 Val Gly Arg Arg Ser Leu Lys Asn Lys Leu Ile Met Trp Asp Pro Asp 385 390 395 400 Arg Tyr Gly Thr Thr Val Pro Tyr Thr Ser Arg Arg Pro Lys Asp Ser 405 410 415 Glu Arg Glu Gly Gln Gly Tyr Ser Phe Val Ser Arg Gly Glu Met Glu 420 425 430 Ala Asp Val Arg Ala Gly Arg Tyr Leu Glu His Gly Glu Tyr Glu Gly 435 440 445 Asn Leu Tyr Gly Thr Arg Ile Asp Ser Ile Arg Gly Val Val Ala Ala 450 455 460 Gly Lys Val Cys Val Leu Asp Val Asn Pro Gln Ala Val Lys Val Leu 465 470 475 480 Arg Thr Ala Glu Phe Val Pro Tyr Val Val Phe Ile Glu Ala Pro Asp 485 490 495 Phe Glu Thr Leu Arg Ala Met Asn Arg Ala Ala Leu Glu Ser Gly Ile 500 505 510 Ser Thr Lys Gln Leu Thr Glu Ala Asp Leu Arg Arg Thr Val Glu Glu 515 520 525 Ser Ser Arg Ile Gln Arg Gly Tyr Gly His Tyr Phe Asp Leu Cys Leu 530 535 540 Val Asn Ser Asn Leu Glu Arg Thr Phe Arg Glu Leu Gln Thr Ala Met 545 550 555 560 Glu Lys Leu Arg Thr Glu Pro Gln Trp Val Pro Val Ser Trp Val Tyr 565 570 575 5 576 PRT Human 5 Met Pro Val Ala Ala Thr Asn Ser Glu Thr Ala Met Gln Gln Val Leu 1 5 10 15 Asp Asn Leu Gly Ser Leu Pro Ser Ala Thr Gly Ala Ala Glu Leu Asp 20 25 30 Leu Ile Phe Leu Arg Gly Ile Met Glu Ser Pro Ile Val Arg Ser Leu 35 40 45 Ala Lys Val Ile Met Val Leu Trp Phe Met Gln Gln Asn Val Phe Val 50 55 60 Pro Met Lys Tyr Met Leu Lys Tyr Phe Gly Ala His Glu Arg Leu Glu 65 70 75 80 Glu Thr Lys Leu Glu Ala Val Arg Asp Asn Asn Leu Glu Leu Val Gln 85 90 95 Glu Ile Leu Arg Asp Leu Ala Gln Leu Ala Glu Gln Ser Ser Thr Ala 100 105 110 Ala Glu Leu Ala His Ile Leu Gln Glu Pro His Phe Gln Ser Leu Leu 115 120 125 Glu Thr His Asp Ser Val Ala Ser Lys Thr Tyr Glu Thr Pro Pro Pro 130 135 140 Ser Pro Gly Leu Asp Pro Thr Phe Ser Asn Gln Pro Val Pro Pro Asp 145 150 155 160 Ala Val Arg Met Val Gly Ile Arg Lys Thr Ala Gly Glu His Leu Gly 165 170 175 Val Thr Phe Arg Val Glu Gly Gly Glu Leu Val Ile Ala Arg Ile Leu 180 185 190 His Gly Gly Met Val Ala Gln Gln Gly Leu Leu His Val Gly Asp Ile 195 200 205 Ile Lys Glu Val Asn Gly Gln Pro Val Gly Ser Asp Pro Arg Ala Leu 210 215 220 Gln Glu Leu Leu Arg Asn Ala Ser Gly Ser Val Ile Leu Lys Ile Leu 225 230 235 240 Pro Asn Tyr Gln Glu Pro His Leu Pro Arg Gln Val Phe Val Lys Cys 245 250 255 His Phe Asp Tyr Asp Pro Ala Arg Asp Ser Leu Ile Pro Cys Lys Glu 260 265 270 Ala Gly Leu Arg Phe Asn Ala Gly Asp Leu Leu Gln Ile Val Asn Gln 275 280 285 Asp Asp Ala Asn Trp Trp Gln Ala Cys His Val Glu Gly Gly Ser Ala 290 295 300 Gly Leu Ile Pro Ser Gln Leu Leu Glu Glu Lys Arg Lys Ala Phe Val 305 310 315 320 Lys Arg Asp Leu Glu Leu Thr Pro Asn Ser Gly Thr Leu Cys Gly Ser 325 330 335 Leu Ser Gly Lys Lys Lys Lys Arg Met Met Tyr Leu Thr Thr Lys Asn 340 345 350 Ala Glu Phe Asp Arg His Glu Leu Leu Ile Tyr Glu Glu Val Ala Arg 355 360 365 Met Pro Pro Phe Arg Arg Lys Thr Leu Val Leu Ile Gly Ala Gln Gly 370 375 380 Val Gly Arg Arg Ser Leu Lys Asn Lys Leu Ile Met Trp Asp Pro Asp 385 390 395 400 Arg Tyr Gly Thr Thr Val Pro Tyr Thr Ser Arg Arg Pro Lys Asp Ser 405 410 415 Glu Arg Glu Gly Gln Gly Tyr Ser Phe Val Ser Arg Gly Glu Met Glu 420 425 430 Ala Asp Val Arg Ala Gly Arg Tyr Leu Glu His Gly Glu Tyr Glu Gly 435 440 445 Asn Leu Tyr Gly Thr Arg Ile Asp Ser Ile Arg Gly Val Val Ala Ala 450 455 460 Gly Lys Val Cys Val Leu Asp Val Asn Pro Gln Ala Val Lys Val Leu 465 470 475 480 Arg Thr Ala Glu Phe Val Pro Tyr Val Val Phe Ile Glu Ala Pro Asp 485 490 495 Phe Glu Thr Leu Arg Ala Met Asn Arg Ala Ala Leu Glu Ser Gly Ile 500 505 510 Ser Thr Lys Gln Leu Thr Glu Ala Asp Leu Arg Arg Thr Val Glu Glu 515 520 525 Ser Ser Arg Ile Gln Arg Gly Tyr Gly His Tyr Phe Asp Leu Cys Leu 530 535 540 Val Asn Ser Asn Leu Glu Arg Thr Phe Arg Glu Leu Gln Thr Ala Met 545 550 555 560 Glu Lys Leu Arg Thr Glu Pro Gln Trp Val Pro Val Ser Trp Val Tyr 565 570 575 6 552 PRT Mus Musculus 6 Met Pro Val Ala Ala Thr Asn Ser Glu Ser Ala Met Gln Gln Val Leu 1 5 10 15 Asp Asn Leu Gly Ser Leu Pro Asn Ala Thr Gly Ala Ala Glu Leu Asp 20 25 30 Leu Ile Phe Leu Arg Gly Ile Met Glu Ser Pro Ile Val Arg Ser Leu 35 40 45 Ala Lys Ala His Glu Arg Leu Glu Glu Thr Lys Leu Glu Ala Val Arg 50 55 60 Asp Asn Asn Leu Glu Leu Val Gln Glu Ile Leu Arg Asp Leu Ala Glu 65 70 75 80 Leu Ala Glu Gln Ser Ser Thr Ala Ala Glu Leu Ala Arg Ile Leu Gln 85 90 95 Glu Pro His Phe Gln Ser Leu Leu Glu Thr His Asp Ser Val Ala Ser 100 105 110 Lys Thr Tyr Glu Thr Pro Pro Pro Ser Pro Gly Leu Asp Pro Thr Phe 115 120 125 Ser Asn Gln Pro Val Pro Pro Asp Ala Val Arg Met Val Gly Ile Arg 130 135 140 Lys Thr Ala Gly Glu His Leu Gly Val Thr Phe Arg Val Glu Gly Gly 145 150 155 160 Glu Leu Val Ile Ala Arg Ile Leu His Gly Gly Met Val Ala Gln Gln 165 170 175 Gly Leu Leu His Val Gly Asp Ile Ile Lys Glu Val Asn Gly Gln Pro 180 185 190 Val Gly Ser Asp Pro Arg Ala Leu Gln Glu Leu Leu Arg Ser Ala Ser 195 200 205 Gly Ser Val Ile Leu Lys Ile Leu Pro Ser Tyr Gln Glu Pro His Leu 210 215 220 Pro Arg Gln Val Phe Val Lys Cys His Phe Asp Tyr Asp Pro Ala Arg 225 230 235 240 Asp Ser Leu Ser Pro Cys Lys Glu Ala Gly Leu Arg Phe Asn Ala Gly 245 250 255 Asp Leu Leu Gln Ile Val Asn Gln Asp Asp Ala Asn Trp Trp Gln Ala 260 265 270 Cys His Val Glu Gly Gly Ser Ala Gly Leu Ile Pro Ser Gln Leu Leu 275 280 285 Glu Glu Lys Arg Lys Ala Phe Val Lys Arg Asp Leu Glu Leu Thr Pro 290 295 300 Thr Ser Gly Thr Leu Cys Gly Ser Leu Ser Gly Lys Lys Lys Lys Arg 305 310 315 320 Met Met Tyr Leu Thr Thr Lys Asn Ala Glu Phe Asp Arg His Glu Leu 325 330 335 Leu Ile Tyr Glu Glu Val Ala Arg Met Pro Pro Phe Arg Arg Lys Thr 340 345 350 Leu Val Leu Ile Gly Ala Gln Gly Val Gly Arg Arg Ser Leu Lys Asn 355 360 365 Lys Leu Ile Leu Trp Asp Pro Asp Arg Tyr Gly Thr Thr Val Pro Tyr 370 375 380 Thr Ser Arg Arg Pro Lys Asp Ser Glu Arg Glu Gly Gln Gly Tyr Ser 385 390 395 400 Phe Val Ser Arg Gly Glu Met Glu Ala Asp Ile Arg Ala Gly Arg Tyr 405 410 415 Leu Glu His Gly Glu Tyr Glu Gly Asn Leu Tyr Gly Thr Arg Ile Asp 420 425 430 Ser Ile Arg Gly Val Val Ala Ser Gly Lys Val Cys Val Leu Asp Val 435 440 445 Asn Pro Gln Ala Val Lys Val Leu Arg Thr Ala Glu Phe Val Pro Tyr 450 455 460 Val Val Phe Ile Glu Ala Pro Asp Tyr Glu Thr Leu Arg Ala Met Asn 465 470 475 480 Arg Ala Ala Leu Glu Ser Gly Val Ser Thr Lys Gln Leu Thr Glu Ala 485 490 495 Asp Leu Arg Arg Thr Val Glu Glu Ser Ser Arg Ile Gln Arg Gly Tyr 500 505 510 Gly His Tyr Phe Asp Leu Ser Leu Val Asn Ser Asn Leu Glu Arg Thr 515 520 525 Phe Arg Glu Leu Gln Thr Ala Met Glu Lys Leu Arg Thr Glu Pro Gln 530 535 540 Trp Val Pro Val Ser Trp Val Tyr 545 550 7 574 PRT Human 7 Gly Cys Ser Ala Ala Pro Val Ala Glu Gly Glu Gln Arg Arg Arg Gly 1 5 10 15 Ala Thr Gly Ser Gly Gly Ser Gly Gly Ala Glu Ala Ala Glu Val Arg 20 25 30 Ala Ala Met Gln Gln Val Leu Glu Asn Leu Thr Glu Leu Pro Ser Ser 35 40 45 Thr Gly Ala Glu Glu Ile Asp Leu Ile Phe Leu Lys Gly Ile Met Glu 50 55 60 Asn Pro Ile Val Lys Ser Leu Ala Lys Ala His Glu Arg Leu Glu Asp 65 70 75 80 Ser Lys Leu Glu Ala Val Ser Asp Asn Asn Leu Glu Leu Val Asn Glu 85 90 95 Ile Leu Glu Asp Ile Thr Pro Leu Ile Asn Val Asp Glu Asn Val Ala 100 105 110 Glu Leu Val Gly Ile Leu Lys Glu Pro His Phe Gln Ser Leu Leu Glu 115 120 125 Ala His Asp Ile Val Ala Ser Lys Cys Tyr Asp Ser Pro Pro Ser Ser 130 135 140 Pro Glu Met Asn Asn Ser Ser Ile Asn Asn Gln Leu Leu Pro Val Asp 145 150 155 160 Ala Ile Arg Ile Leu Gly Ile His Lys Arg Ala Gly Glu Pro Leu Gly 165 170 175 Val Thr Phe Arg Val Glu Asn Asn Asp Leu Val Ile Ala Arg Ile Leu 180 185 190 His Gly Gly Met Ile Asp Arg Gln Gly Leu Leu His Val Gly Asp Ile 195 200 205 Ile Lys Glu Val Asn Gly His Glu Val Gly Asn Asn Pro Lys Glu Leu 210 215 220 Gln Glu Leu Leu Lys Asn Ile Ser Gly Ser Val Thr Leu Lys Ile Leu 225 230 235 240 Pro Ser Tyr Arg Asp Thr Ile Thr Pro Gln Gln Val Phe Val Lys Cys 245 250 255 His Phe Asp Tyr Asn Pro Tyr Asn Asp Asn Leu Ile Pro Cys Lys Glu 260 265 270 Ala Gly Leu Lys Phe Ser Lys Gly Glu Ile Leu Gln Ile Val Asn Arg 275 280 285 Glu Asp Pro Asn Trp Trp Gln Ala Ser His Val Lys Glu Gly Gly Ser 290 295 300 Ala Gly Leu Ile Pro Ser Gln Phe Leu Glu Glu Lys Arg Lys Ala Phe 305 310 315 320 Val Arg Arg Asp Trp Asp Asn Ser Gly Pro Phe Cys Gly Thr Ile Ser 325 330 335 Ser Lys Lys Lys Lys Lys Met Met Tyr Leu Thr Thr Arg Asn Ala Glu 340 345 350 Phe Asp Arg His Glu Ile Gln Ile Tyr Glu Glu Val Ala Lys Met Pro 355 360 365 Pro Phe Gln Arg Lys Thr Leu Val Leu Ile Gly Ala Gln Gly Val Gly 370 375 380 Arg Arg Ser Leu Lys Asn Arg Phe Ile Val Leu Asn Pro Thr Arg Phe 385 390 395 400 Gly Thr Thr Val Pro Phe Thr Ser Arg Lys Pro Arg Glu Asp Glu Lys 405 410 415 Asp Gly Gln Ala Tyr Lys Phe Val Ser Arg Ser Glu Met Glu Ala Asp 420 425 430 Ile Lys Ala Gly Lys Tyr Leu Glu His Gly Glu Tyr Glu Gly Asn Leu 435 440 445 Tyr Gly Thr Lys Ile Asp Ser Ile Leu Glu Val Val Gln Thr Gly Arg 450 455 460 Thr Cys Ile Leu Asp Val Asn Pro Gln Ala Leu Lys Val Leu Arg Thr 465 470 475 480 Ser Glu Phe Met Pro Tyr Val Val Phe Ile Ala Ala Pro Glu Leu Glu 485 490 495 Thr Leu Arg Ala Met His Lys Ala Val Val Asp Ala Gly Ile Thr Thr 500 505 510 Lys Leu Leu Thr Asp Ser Asp Leu Lys Lys Thr Val Asp Glu Ser Ala 515 520 525 Arg Ile Gln Arg Ala Tyr Asn His Tyr Phe Asp Leu Ile Ile Ile Asn 530 535 540 Asp Asn Leu Asp Lys Ala Phe Glu Lys Leu Gln Thr Ala Ile Glu Lys 545 550 555 560 Leu Arg Met Glu Pro Gln Trp Val Pro Ile Ser Trp Val Tyr 565 570 8 539 PRT Mus Musculus 8 Met Gln Gln Val Leu Glu Asn Leu Thr Glu Leu Pro Ser Ser Thr Gly 1 5 10 15 Ala Glu Glu Ile Asp Leu Ile Phe Leu Lys Gly Ile Met Glu Asn Pro 20 25 30 Ile Val Lys Ser Leu Ala Lys Ala His Glu Arg Leu Glu Asp Ser Lys 35 40 45 Leu Glu Ala Val Ser Asp Asn Asn Leu Glu Leu Val Asn Glu Ile Leu 50 55 60 Glu Asp Ile Thr Pro Leu Ile Ser Val Asp Glu Asn Val Ala Glu Leu 65 70 75 80 Val Gly Ile Leu Lys Glu Pro His Phe Gln Ser Leu Leu Glu Ala His 85 90 95 Asp Ile Val Ala Ser Lys Cys Tyr Asp Ser Pro Pro Ser Ser Pro Glu 100 105 110 Met Asn Ile Pro Ser Leu Asn Asn Gln Leu Pro Val Asp Ala Ile Arg 115 120 125 Ile Leu Gly Ile His Lys Lys Ala Gly Glu Pro Leu Gly Val Thr Phe 130 135 140 Arg Val Glu Asn Asn Asp Leu Val Ile Ala Arg Ile Leu His Gly Gly 145 150 155 160 Met Ile Asp Arg Gln Gly Leu Leu His Val Gly Asp Ile Ile Lys Glu 165 170 175 Val Asn Gly His Glu Val Gly Asn Asn Pro Lys Glu Leu Gln Glu Leu 180 185 190 Leu Lys Asn Ile Ser Gly Ser Val Thr Leu Lys Ile Leu Pro Ser Tyr 195 200 205 Arg Asp Thr Ile Thr Pro Gln Gln Val Phe Val Lys Cys His Phe Asp 210 215 220 Tyr Asn Pro Phe Asn Asp Asn Leu Ile Pro Cys Lys Glu Ala Gly Leu 225 230 235 240 Lys Phe Ser Lys Gly Glu Ile Leu Gln Ile Val Asn Arg Glu Asp Pro 245 250 255 Asn Trp Trp Gln Ala Ser His Val Lys Glu Gly Gly Ser Ala Gly Leu 260 265 270 Ile Pro Ser Gln Phe Leu Glu Glu Lys Arg Lys Ala Phe Val Arg Arg 275 280 285 Asp Trp Asp Asn Ser Gly Pro Phe Cys Gly Thr Ile Ser Asn Lys Lys 290 295 300 Lys Lys Lys Met Met Tyr Leu Thr Thr Arg Asn Ala Glu Phe Asp Arg 305 310 315 320 His Glu Ile Gln Ile Tyr Glu Glu Val Ala Lys Met Pro Pro Phe Gln 325 330 335 Arg Lys Thr Leu Val Leu Ile Gly Ala Gln Gly Val Gly Arg Arg Ser 340 345 350 Leu Lys Asn Arg Phe Ile Val Leu Asn Pro Ala Arg Phe Gly Thr Thr 355 360 365 Val Pro Phe Thr Ser Arg Lys Pro Arg Glu Asp Glu Lys Asp Gly Gln 370 375 380 Ala Tyr Lys Phe Val Ser Arg Ser Glu Met Glu Ala Asp Ile Lys Ala 385 390 395 400 Gly Lys Tyr Leu Glu His Gly Glu Tyr Glu Gly Asn Leu Tyr Gly Thr 405 410 415 Lys Ile Asp Ser Ile Leu Glu Val Val Gln Thr Gly Arg Thr Cys Ile 420 425 430 Leu Asp Val Asn Pro Gln Ala Leu Lys Val Leu Arg Thr Ser Glu Phe 435 440 445 Met Pro Tyr Val Val Phe Ile Ala Ala Pro Glu Leu Glu Thr Leu Arg 450 455 460 Ala Met His Lys Ala Val Val Asp Ala Gly Ile Thr Thr Lys Leu Leu 465 470 475 480 Thr Asp Ser Asp Leu Lys Lys Thr Val Asp Glu Ser Ala Arg Ile Gln 485 490 495 Arg Ala Tyr Asn His Tyr Phe Asp Leu Ile Ile Val Asn Asp Asn Leu 500 505 510 Asp Lys Ala Phe Glu Lys Leu Gln Thr Ala Ile Glu Lys Leu Arg Met 515 520 525 Glu Pro Gln Trp Val Pro Ile Ser Trp Val Tyr 530 535 

What is claimed is:
 1. An isolated nucleic acid molecule encoding a membrane-associated guanylate kinase protein consisting of a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence that encodes a protein comprising the an amino acid sequence of SEQ ID NO:2; (b) a nucleic acid molecule consisting of the nucleic acid sequence of SEQ ID NO:1; and (c) a nucleic acid molecule consisting of the nucleic acid sequence of SEQ ID NO:3.
 2. A nucleic acid vector comprising a nucleic acid molecule of claim
 1. 3. A host cell containing the vector of claim
 2. 4. A process for producing a polypeptide comprising culturing the host cell of claim 3 under conditions sufficient for the production of said polypeptide, and recovering the peptide from the host cell culture.
 5. An isolated polynucleotide consisting of a nucleotide sequence set forth in SEQ ID NO:1.
 6. An isolated polynucleotide consisting of a nucleotide sequence set forth in SEQ ID NO:3.
 7. A vector according to claim 2, wherein said vector is selected from the group consisting of a plasmid, virus, and bacteriophage.
 8. A vector according to claim 2, wherein said isolated nucleic acid molecule is inserted into said vector in proper orientation and correct reading frame such that the protein of SEQ ID NO:2 may be expressed by a cell transformed with said vector.
 9. A vector according to claim 8, wherein said isolated nucleic acid molecule is operatively linked to a promoter sequence.
 10. An isolated nucleic acid molecule consisting of a nucleotide sequence that is completely complementary to a nucleotide sequence of claim
 1. 